DNA encoding tumor necrosis factor stimulated gene 6 (TSG-6)

ABSTRACT

TSG protein and functional derivatives thereof, DNA coding therefor, expression vehicles, such as plasmids, and host cells transformed or transfected with the DNA molecule, and methods for producing the protein and the DNA are provided, as well as antibodies specific for the TSG-6 protein; a method for detecting the presence of TSG-6 protein in a biological sample; a method for detecting the presence of nucleic acid encoding a normal or mutant TSG-6 protein; a method for measuring induction of expression of TSG-6 in a cell using either nucleic acid hybridization or immunoassay; a method for identifying a compound capable of inducing the expression of TSG-6 in a cell; and a method for measuring the ability of a cell to respond to TNF.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a division of application Ser. No.09/206,695, filed Dec. 7, 1998, which is a division of application Ser.No. 08/242,097, filed May 13, 1994, now issued as U.S. Pat. No.5,846,763, which is a continuation-in-part of application Ser. No.08/024,868, filed Mar. 1, 1993, now issued as U.S. Pat. No. 5,386,013,which is a continuation of application Ser. No. 07/642,312, filed Jan.14, 1991, now abandoned, the entire contents of said applications beingentirely incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to a protein, TSG-6, inducible inconnective tissue cells by tumor necrosis factor or interleukin-1, DNAand mRNA encoding the TSG-6 protein, functional derivatives of theprotein, antibodies specific to the protein, methods of producing theprotein and DNA, and uses of the protein, DNA, mRNA, peptides andantibodies.

[0004] 2. Description of the Background Art

[0005] Tumor necrosis factor (TNF) is a powerful pleiotropic cytokineimportant in host defenses against tumors and infectious agents. TNF hasalso been implicated in the pathology of some neoplastic diseases,infections and autoimmune disorders. Most biological actions of TNF canbe attributed to the triggering of complex genetic programs in thetarget cells. Several genes activated by TNF have been identified butmany more require characterization.

[0006] General Properties of TNF

[0007] TNF (also termed TNF-α and cachectin) is a protein produced byactivated monocytes/macrophages which was originally detected in theserum of animals injected sequentially with a bacterial vaccine(bacillus Calmette-Guerin, BCG) and endotoxin (Carswell, E. A. et al,Proc. Natl. Acad. Sci. USA 72:3666 (1975)). TNF is structurally andfunctionally related to a cytokine produced by activated T lymphocyteswhich was originally termed lymphotoxin (LT) and is also known as TNF-β(Aggarwal, B. B. et al, J. Biol. Chem. 260:2334 (1985); Williams, T. W.et al, Nature 219:1076 (1968); Ruddle, N. H. et al, J. Exp. Med.128:1267 (1968); Spies, T. et al, Proc. Natl. Acad. Sci. USA 83:8699(1986); Gray, P. W. et al, Nature 312:721 (1984); Pennica, D. W. et al,Nature 312:724 (1984)). The genes encoding TNF and LT are linked, andare near the HLA-DR locus on the short arm of human chromosome 6 (Spies,T. et al, supra). TNF and LT bind to common cell surface receptors(Aggarwal, B. B. et al, Nature 318:665 (1985)).

[0008] Natural human TNF is a 157 amino acid, non-glycosylated proteinwith a molecular weight of approximately 17 kDa under denaturingconditions. The mature molecule is derived from a precursor (pre-TNF)which contains 76 additional amino acids at the N-terminus (Pennica, D.W. et al, supra). The expression of the gene encoding TNF is not limitedto cells of the monocyte/macrophage family. Several human non-monocytictumor cell lines were shown to produce TNF (Rubin, B. Y. et al, J. Exp.Med. 164:1350 (1986); Spriggs, D. et al, Proc. Natl. Acad. Sci. USA84:6563 (1987)). TNF is also produced by CD4⁺ and CD8⁺ peripheral bloodT lymphocytes, and by various cultured T and B cell lines (Cuturi, M.C., et al, J. Exp. Med. 165:1581 (1987); Sung, S.-S. J. et al, J. Exp.Med. 168:1539 (1988)).

[0009] Accumulating evidence indicates that TNF is a regulatory cytokinewith pleiotropic biological activities. These activities include:inhibition of lipoprotein lipase synthesis (“cachectin” activity)(Beutler, B. et al, Nature 316:552 (1985)), activation ofpolymorphonuclear leukocytes (Klebanoff, S. J. et al, J. Immunol.136:4220 (1986); Perussia, B., et al, J. Immunol. 138:765 (1987)),inhibition of cell growth or stimulation of cell growth (Vilcek, J. etal, J. Exp. Med. 163:632 (1986); Sugarman, B. J. et al, Science 230:943(1985); Lachman, L. B. et al, J. Immunol. 138:2913 (1987)), cytotoxicaction on certain transformed cell types (Lachman, L. B. et al, supra;Darzynkiewicz, Z. et al, Canc. Res. 44:83 (1984)), antiviral activity(Kohase, M. et al, Cell 45:659 (1986); Wong, G. H. W. et al, Nature323:819 (1986)), stimulation of bone resorption (Bertolini, D. R. et al,Nature 319:516 (1986); Saklatvala, J., Nature 322:547 (1986)),stimulation of collagenase and prostaglandin E2 production (Dayer, J.-M.et al, J. Exp. Med. 162:2163 (1985)), and other actions. For reviews ofTNF, see Beutler, B. et al, Nature 320:584 (1986), Old, L. J., Science230:630 (1986), and Le, J. et al, Lab. Invest. 56:234 (1987).

[0010] TNF also has immunoregulatory actions, including activation of Tcells (Yokota, S. et al, J. Immunol. 140:531 (1988)), B cells (Kehrl, J.H. et al, J. Exp. Med. 166:786 (1987)), monocytes (Philip, R. et al,Nature 323:86 (1986)), thymocytes (Ranges, G. E. et al, J. Exp. Med.167:1472 (1988)), and stimulation of the cell-surface expression ofmajor histocompatibility complex (MHC) class I and class II molecules(Collins, T. et al, Proc. Natl. Acad. Sci. USA 83:446 (1986);Pujol-Borrell, R. et al, Nature 326:304 (1987)).

[0011] TNF also has various pro-inflammatory actions which result intissue injury, such as induction of procoagulant activity on vascularendothelial cells (Pober, J. S. et al, J. Immunol. 136, 1680, 1986)),increased adherence of neutrophils and lymphocytes (Pober, J. S. et al,J. Immunol. 138:3319 (1987)), and stimulation of the release of plateletactivating factor (PAF) from macrophages, neutrophils and vascularendothelial cells (Camussi, G. et al, J. Exp. Med. 166:1390 (1987)).Recent evidence implicates TNF in the pathogenesis of many infections(Cerami, A. et al, Immunol. Today 9:28 (1988)), immune disorders(Piguet, P.-F. et al, J. Exp. Med. 166:1280 (1987)), and in cachexiaaccompanying some malignancies (Oliff, A. et al, Cell 50:555 (1987)).Michie, H. R. et al, Br. J. Surg. 76:670-671 (1989), reviewed evidencethat TNF is the principal mediator associated with the pathologicalchanges of severe sepsis.

[0012] TNF also has activity associated with growth and differentiationof hemopoietic precursor cells (Murphy, M. et al, J. Exp. Med. 164:263(1986); Broxmeyer, H. E. et al, J. Immunol. 136:4487 (1986)); some ofthese actions may be indirect, and are thought to be mediated throughthe stimulation of production of granulocyte-macrophage colonystimulating factor (GM-CSF) (Munker, R. et al, Nature 323:79 (1986)) andother hemopoietic growth factors (Zucali, J. R. et al, J. Immunol.140:840 (1988)).

[0013] Regulation of Gene Expression by TNF

[0014] It is, therefore, apparent that TNF is an extremely “versatile”and clinically significant cytokine. Most of its actions are likely tobe mediated by the activation or inactivation of specific genes in thecells upon which it acts. One exception to this mode of action is therapid cytotoxic effect of TNF on certain target cells; this effect isaugmented by inhibitors of RNA or protein synthesis and does not appearto depend on the modulation of gene expression (Matthews, N., Br. J.Cancer 48:405 (1983)). Many specific gene products have been shown to beup-regulated in TNF-treated cells, some of which are discussed below.

[0015] Among the first examples of TNF-modulated gene expression was thedemonstration that TNF treatment induced an increase in MHC class I mRNAlevels and in surface expression of the MHC class I glycoproteins inhuman vascular endothelial cells (HUVEC) and normal skin fibroblasts(Collins, T. et al, supra). A partial list of other molecules (or genes)induced by TNF appears in Table 1, below. It is interesting to note thatTNF is an autoregulatory cytokine, since exogenously added TNF increasesTNF synthesis in monocytes and monocytic cell lines (Philip, R. et al,Nature 323:86 (1986); Schmid, J. et al, J. Immunol. 139:250 (1987)).TABLE 1 GENES AND PROTEINS INDUCED BY TUMOR NECROSIS FACTOR Protein orGene Cell Type Ref Leukocyte adhesion protein H4/18 HUVEC  (1)Platelet-derived growth factor HUVEC and some tumor cell  (2) (PDGF)lines IL-6 (IFN-β2 or BSF-2) Human skin fibroblasts  (3) HLA-DR Humantumor cell lines  (4) Collagenase Synovial cells and skin  (5)fibroblasts 2′-5′ oligoadenylate synthetase Tumor cell lines  (6) c-mycand c-fos oncogenes Human skin fibroblasts  (7) Epidermal growth factorreceptor Human skin fibroblasts  (8) Tissue factor HUVEC  (9) ICAM-1 andELAM-1 HUVEC (10) Plasminogen activator inhibitors HT1080 cell line (11)1 and 2 (PAI-1 and PAI-2) Synthesis of 36 kDa and 42 kDa Human skinfibrobiasts (12) (= PAI-2) proteins Superoxide Dismutase (MnSOD) Humantumor cell lines (13) gene IL-1α and IL-1β genes Human skin fibroblasts(14)

[0016] The inhibitory actions of TNF on gene expression are lesswell-characterized. TNF was shown to inhibit c-myc expression in cellswhose growth it inhibited (Kronke, M. et al, Proc. Natl. Acad. Sci. USA84:469 (1987)). Collagen synthesis was inhibited in human fibroblasts(Solis-Herruzo et al, J. Biol. Chem. 263:5841 (1988)), andthrombomodulin in HUVEC (Conway, E. M. et al, Molec. Cell. Biol. 8:5588(1988)). All these inhibitory actions were expressed at the level oftranscription, but the precise mechanisms are still unclear.

[0017] The mechanisms of signal transduction and gene activation by TNFare the subject of great interest. In many cell types, TNF activates aphospholipase (most likely PLA2), resulting in the liberation ofarachidonic acid from cellular pools (Suffys, P. et al, Biochem.Biophys. Res. Comm. 149:735 (1987)) and increased eicosanoid synthesis(Dayer, J.-M. et al, supra). In human fibroblasts, TNF stimulated GTPaseactivity (Imamura, K. et al J. Biol. Chem. 263:10247 (1989)), raisedcAMP levels, enhanced cAMP-dependent protein kinase activity, andactivated protein kinase C (PKC) (Zhang, Y. et al, Proc. Natl. Acad.Sci. USA 85:6802 (1988); Brenner, D. A. et al, Nature 337:661 (1989)).TNF can also activate the transcription factor NF-kB, which appears tobe the mechanism by which TNF induces the IL-2 receptor α chain(Lowenthal, J. W. et al, Proc. Natl. Acad. Sci. USA 86:2231 (1989)) orcause activation of latent human immunodeficiency virus, HIV-1 (Griffin,G. E. et al, Nature 339:70 (1989)).

[0018] Interactions of TNF with Other Cytokines

[0019] When the individual actions of TNF-α, TNF-β, IL-1α, IL-1β, IFN-α,IFN-β or IFN-τ are compared in various experimental systems, a greatdeal of apparent redundancy and ambiguity is noted. First, structurallyrelated cytokines which utilize the same receptor (e.g., TNF-α andTNF-β; IL-1α and IL-β; IFN-α and IFN-β) act similarly. Moresurprisingly, structurally unrelated cytokines which bind to differentreceptors also have similar physiological effects. For example, IL-1 andTNF have similar gene activating activities, and result in similarbiological effects (Le, J. et al Lab. Invest. 56:234 (1987)). IFNs andTNF also share biological activities (Kohase, M. et al, Cell 45:659(1986); Wong, G. H. W. et al, Nature 323:819 (1986); Williamson, B. D.et al, Proc. Natl. Acad. Sci. USA 80:5397 (1983); Stone-Wolff, D. S. etal, J. Exp. Med. 159:828 (1984)). For example, IFNs and TNF activatesome of the same genes, including MHC class I and class II genes, 2′-5′oligo-adenylate synthetase, IL-6, the transcription factor IRF-1, andthe TNF gene itself (Vilcek, J., Handbook of Experimental Pharmacology,Vol. 95/II, p. 3, Springer-Verlag, Berlin (1990)).

[0020] Under natural conditions, cells are rarely, if ever, exposed to asingle cytokine. Rather, cytokine action in vivo is “contextual,” as hasbeen postulated for growth factors (Sporn, M. B. et al, Nature 332:217(1988)). The biological effects produced by cytokines under naturalconditions must therefore represent the sum of the synergistic andantagonistic interactions of all cytokines present simultaneously in agiven microenvironment. In addition, cytokines appear to be arranged in“networks” and “cascades”, such that the synthesis of one cytokine canbe positively or negatively regulated by another. For these reasons, itis important to understand the molecular mechanisms of action ofcytokines acting individually as well as in combination.

[0021] In contrast to the above, there are cases in which the actions ofTNF and IFNs are antagonistic rather than similar or synergistic. Forexample, TNF is mitogenic for human diploid fibroblasts, whereas IFNsinhibit growth of these cells (Vilcek, J. et al, J. Exp. Med. 163:632(1986)). The cellular response to a combination of TNF and an IFN candiffer from the response to either one alone, both qualitatively andquantitatively (Leeuwenberg, J. F. M. et al, J. Exp. Med. 166:1180(1987); Reis, L. F. L. et al, J. Biol. Chem. 264:16351 (1989); Feinman,R. et al, J. Immunol. 136:2441 (1986); Trinchieri, G. et al, Abstr. 2ndInt'l Conf. TNF, p. 7 (1989)). To make matters even more complicated, insome cells TNF can induce IFN-β synthesis (Reis et al, supra); theactivation of some genes (e.g., HLA class I) by TNF requires thepresence of IFN-β (Leeuwenberg et al, supra). Since IFNs and TNF-α andTNF-β are often produced in the same microenvironment in response to asimilar set of stimuli (Murphy, M. et al, supra; Stone-Wolff et al,supra; Billiau, A., Immunol. Today 9:37 (1988)), it is clear that theinteractions of TNF and IFNs are highly relevant to the outcome in vivounder either “normal” or pathophysiological conditions.

[0022] The association of cytokines, in particular TNF, with cancer andinfectious diseases takes many forms often related to the host'scatabolic state. One of the major and most characteristic problems seenin cancer patients is weight loss, usually associated with anorexia. Theextensive wasting which results is known as “cachexia” (see, for review,Kern, K. A. et al (J. Parent. Enter. Nutr. 12:286-298 (1988)). Cachexiaincludes progressive weight loss, anorexia, and persistent erosion ofbody mass in response to a malignant growth. The fundamentalphysiological derangement may be related to a decline in food intakerelative to energy expenditure. The causes for this commonly observedand often life-limiting disturbance remain to be determined, even thoughmany contributing factors have been identified (Braunwald, E. et al(Eds.), Harrison's Principles of Internal Medicine, 11th Ed.,McGraw-Hill Book Co., New York, 1987, Chap. 78, pp. 421-431). Thecachectic state is associated with significant morbidity and isresponsible for the majority of cancer mortality. A number of studieshave suggested that TNF is an important mediator of the cachexia incancer, infectious disease, and in other catabolic states.

[0023] It has been known for some time that in bacterial infection,sepsis and critical illness, bacterial lipopolysaccharides (LPS), orendotoxins, are responsible for many of the pathophysiologicalmanifestations, including fever, malaise, anorexia, and cachexia. Morerecently, it was observed that TNF can mimic many endotoxin effects,leading to the suggestion that TNF, and related cytokines derived fromcells of the macrophage/monocyte family, in particular, IL-1, arecentral mediators responsible for the clinical manifestations of theillness. Endotoxin is a potent monocyte/macrophage activator whichstimulates production and secretion of TNF (Kornbluth, S. K. et al, J.Immunol. 137:2585-2591 (1986)) and other cytokines including IL-1(Dinarello, C. A., Rev. Infec. Dis. 6:51-94 (1984)), interleukin-6(IL6), and colony stimulating factor (CSF) (Apte, R. N. et al J. Cell.Physiol. 89:313 (1976)). Some of these cytokines further stimulate Tlymphocytes to produce additional cytokines, for example, interleukin-2(IL-2) (Robb, R. J., Immunol. Today 5:203-209 (1984)).

[0024] The monocyte-derived cytokines are thought to be importantmediators of the metabolic and neurohormonal responses to endotoxin(Michie, H. R. et al, N. Eng. J. Med. 318:1481-1486 (1988)), and incancer and other catabolic states (Norton, J. A. et al, Nutrition5:131-135 (1989)). Interestingly, some changes induced by low-dose TNFclosely resemble changes provoked by high dose IL2 (Remick, D. G. et al,Lab. Invest. 56:583-590 (1987)).

[0025] Endotoxin administration to human volunteers produced acuteillness with flu-like symptoms including fever, tachycardia, increasedmetabolic rate and stress hormone release (Revhaug, A. et al, Arch.Surg. 123:162-170 (1988)). Treatment of cancer patients (having normalkidney and liver function) with escalating doses of TNF (4-636 μg/m²/24hr) indicated that doses greater than 545 μg/m²/24 hr caused alterationssimilar to those induced by injection of endotoxin (4 ng/kg) intohealthy humans (Michie, H. R. et al, Surgery 104:280-286 (1988)),leading the authors to conclude that TNF is the principal host mediatorof septic and endotoxemic responses. More recently, it was shown thatfive days of chronic intravenous TNF infusion into humans or rats wasassociated with anorexia, fluid retention, acute phase responses, andnegative nitrogen balance (i.e., classic catabolic effects), leading tothe conclusion that TNF may be responsible for many of the changes notedduring critical illness (Michie, H. R. et al, Ann. Surg. 209:19-24(1989)). Administration of rTNF to cancer patients also led to a rise inC-reactive protein (CRP) and a fall in serum zinc, a large increase inforearm efflux of total amino acids, and amino acid uptake by othertissues (Warren, R. S. et al, Arch. Surg. 122:1396-1400 (1987)),considered further evidence for a role of TNF in cancer cachexia.

[0026] Citation of any document herein is not intended as an admissionthat such document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

[0027] Cytokines such as TNF and IL-1 play a major role in the mediationof inflammatory responses as well as in host responses to infections andcancer. The mode of action of these cytokines is only beginning to beunderstood. The present inventors have discovered and studied a seriesof proteins and glycoproteins induced in connective tissue cells by suchcytokines. As a result of these studies, the present inventors haveconceived of the use of such cytokine-induced proteins or glycoproteins,termed TSG proteins, or functional derivatives such as peptides derivedtherefrom, and antibodies specific for these TSG proteins/glycoproteins,for a number of diagnostic and therapeutic procedures. These proteins,the DNA coding therefor, and the functional derivatives thereof, areuseful in a number of diseases associated with action of the above typesof cytokines, including chronic inflammatory conditions, in particularrheumatoid arthritis, in infections and sepsis, and in cancer.

[0028] Specifically, the present invention provides a cytokine-inducedprotein or glycoprotein molecule, termed TSG-6, or a functionalderivative thereof, wherein, when the protein molecule is one whichnaturally occurs, it is substantially free of other proteins orglycoproteins with which it is natively associated. The full lengthprotein molecule has an apparent molecular weight of about 32 kDa or 35kDa and has the amino acid sequence SEQ ID NO:2 or as presented in Table2. In a glycosylated form, the glycoprotein may have molecular weightsin the range of about 35 kDa or 38-41 kDa.

[0029] The present invention is further directed to a DNA moleculeencoding TSG-6 or a functional derivative thereof, wherein, when the DNAmolecule occurs naturally, it is substantially free of other nucleotidesequences with which it is natively associated, in particular itsadjoining sequences. In a preferred embodiment, the DNA molecule has thenucleotide sequence SEQ ID NO:1. The DNA molecule of the presentinvention may be genomic DNA or cDNA and it may be single stranded ordouble stranded.

[0030] The present invention provides the DNA molecule as an expressionvehicle, such as a plasmid, and provides host cells transformed ortransfected with the DNA molecule. Hosts may be bacteria or eukaryoticcells, including yeast and mammalian cells.

[0031] Also included in the present invention is a process for preparingthe TSG-6 protein or glycoprotein molecule substantially free of otherproteins or glycoproteins with which it is natively associated, or afunctional derivative thereof, comprising: (a) culturing a host cellcapable of expressing the protein under culturing conditions, (b)expressing the protein or functional derivative; and (c) recovering theprotein or functional derivative from the culture.

[0032] The present invention is further directed to an antibody specificfor the TSG-6 protein or an epitope thereof. A preferred antibody is amonoclonal antibody.

[0033] Also provided is a method for detecting the presence of TSG-6protein in a biological sample, comprising:

[0034] (a) contacting the biological sample that is suspected ofcontaining TSG-6 protein with a molecule capable of binding to theprotein; and

[0035] (b) detecting any of this molecule bound to the protein.

[0036] For this method, a preferred molecule is an antibody or antibodyfragment, most preferably a monoclonal antibody, and the preferreddetection method is an immunoassay.

[0037] The present invention further includes a method for detecting thepresence of nucleic acid encoding a normal or mutant TSG-6 protein in asubject comprising: (a) contacting a cell obtained from the subject, anextract thereof, or a culture supernatant thereof, with anoligonucleotide probe encoding at least a portion of the normal ormutant TSG-6 under hybridizing conditions; and (b) measuring thehybridization of this probe to the nucleic acid of the cell, therebydetecting the presence of the nucleic acid. This method may additionallyinclude, before step (a), selectively amplifying the amount of DNA ofthe cell encoding the TSG-6 protein.

[0038] The present invention is still further directed to a method formeasuring induction of expression of TSG-6 in a cell, comprising: (a)contacting the cell with a substance capable of inducing expression ofTSG-6; (b) measuring the amount of mRNA encoding TSG-6 in the cell byhybridization with an oligonucleotide probe encoding at least a portionof TSG-6, under hybridizing conditions; and (c) comparing the amount ofTSG-6 mRNA in the cell with the amount of TSG-6 mRNA in the cell notcontacted with the inducing substance, wherein an increase in the amountof the TSG-6 mRNA indicates that the induction has occurred.

[0039] An alternative method for measuring induction of expression ofTSG-6, according to the present invention, comprises: (a) contacting thecell with a substance capable of inducing expression of TSG-6; (b)measuring the amount of TSG-6 protein in an extract or supernatant ofthe cell using the method described above for measuring the TSG-6protein, preferably, an immunoassay; (c) comparing the amount of TSG-6protein in the cell extract or supernatant with the amount of TSG-6protein in the extract or supernatant of a cell not contacted with theinducing substance, wherein an increase in the amount of the TSG-6protein indicates that the induction has occurred.

[0040] The present invention may also be used in a method foridentifying a compound capable of inducing the expression of TSG-6 in acell, comprising: (a) contacting the cell with the compound beingtested; and (b) measuring the induction of TSG-6 mRNA according to oneof the two methods described above, thereby identifying the compound.

[0041] The present invention provides a method for measuring the abilityof a cell to respond to TNF or to IL-1, comprising: (a) contacting thecell with an amount of TNF capable of inducing expression of the TSG-6gene in FS-4 cells; and (b) determining the induction of expression ofTSG-6 mRNA or protein using either of the methods described above,thereby measuring the ability of the cell to respond to TNF.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGS. 1A-1H depict Northern blots showing induction of mRNAscorresponding to eight TSG cDNAs in FS-4 cells treated with TNF.Growth-arrested FS-4 cells were exposed to TNF (20 ng/ml) at 0 h. Atdifferent intervals thereafter, total cell RNA was isolated,fractionated on formaldehyde-agarose gels, transferred to Zeta-probeblotting membranes, and hybridized separately to each of the ³²P-labeledTSG cDNA inserts. To ascertain whether equal amounts of RNA were loadedin each lane, most blots were also probed with a ³²P-labeled pHe7internal reference cDNA insert specific for an invariant mRNA species ofabout 1.0 kb.

[0043] FIGS. 2A-2H are a series of graphs showing the kinetics ofinduction of eight TSG mRNAs by TNF. Autoradiograms of the Northernblots shown in FIGS. 1A-1H were scanned by laser densitometry. For eachindividual mRNA, the highest-density band was normalized to represent100% induction.

[0044] FIGS. 3A-3C show the nucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of TSG-6 cDNA. Nucleotide andamino acid residues are numbered from the first methionine of the majoropen reading frame. The putative signal sequence is underlined (thickline). Potential glycosylation sites for N-linked glycans are shown bydouble broken lines. Potential chondroitin sulfate linkage site andconsensus sequence are shown by star with single broken line. Alsomarked are the mRNA decay consensus sequence motifs ATTTA (thin line)(Shaw, G. et al, Cell 46:659 (1986)) and the polyadenylation signal (^ ^^ ^ ^ ) are underscored.

[0045]FIG. 4 is a schematic diagram of the putative secondary structureof the TSG-6 protein. The possible signal peptide sequence, and regionswith homology to cartilage link protein/proteoglycan core/lymphocytehoming receptor CD44, and to C1r A chain are indicated. Also depictedare two potential N-glycosylation sites (ball and stick) and achondroitin sulfate linkage site (asterisk and stick).

[0046] FIGS. 5A-5B show, in FIG. 5A, the alignment of the putative aminoacid sequence of TSG-6 corresponding to amino acid residues 36-130 ofSEQ ID NO:2 with the published sequences of human lymphocyte homingreceptor CD44/Hermes (SEQ ID NO:3), rat cartilage link protein (SEQ IDNO:4) and rat proteoglycan core protein (SEQ ID NO:5). The numbering ofamino acid residues corresponds to the putative TSG-6 protein sequence.Note that cysteines in positions 58, 82, 103 and 127 are conserved inall four sequences. FIG. 5B shows the alignment of the C-terminalportion of TSG-6 (amino acids 136 to 240 of SEQ ID NO:2) with theα-fragment of complement component, C1r (SEQ ID NO:6).

[0047]FIGS. 6A and 6B present schematic diagrams of TSG-6 bacterialexpression vectors. FIG. 6A represents the TrpE/TSG-6 fusion proteinexpression vector, pATH-TSG-6. FIG. 6B represents the MS2/TSG-6 fusionprotein expression vector, pEX-TSG-6.

[0048]FIG. 7 is a gel pattern showing the expression and purification ofTrpE/TSG-6 bacterial fusion protein. E. coli HB101 cells transformedwith either pATH-21 or pATH-TSG-6 were induced by 3-β-indole acrylicacid. Total cell lysates were analyzed by SDS-PAGE (10%) and proteinsstained with Coomassie blue. Lane 1: total cell extract, after 24 hrinduction, of cells transformed with pATH-21; Lanes 2 and 3: total cellextract after 3 hr (lane 2) or 24 hr (lane 3) induction of cellstransformed with pPATH-TSG-6. A 7M urea extract of the insolubleproteins of the bacteria shown in lane 3 was fractionated by preparativeSDS-PAGE. TrpE/TSG-6 fusion protein was purified by two rounds ofelectroelution and analyzed by SDS-PAGE (10%). Lane 4: the first eluate(20 μg); Lane 5, 6 and 7: the second eluate (50 μg, 20 μg and 5 μg,respectively); Lane M: marker protein with MW indicated in kDa.

[0049]FIG. 8 is a gel pattern showing expression and purification ofMS2/TSG-6 bacterial fusion protein. E. coli K12 *H*Trp cells transformedwith pEX-TSG-6 were induced by high temperature (42° C.). Total lysatesfrom non-induced (28° C.) and induced (42° C.) cells were analyzed as inFIG. 7. Lane 1: total cell lysate before induction; Lane 2: total celllysate after induction. Lane 3-6: Electroeluted fusion protein from gelslice of preparative SDS-PAGE (5, 10, 20 and 50 μg, respectively).

[0050]FIGS. 9A and 9B are a schematic diagram of TSG-6 expressionvectors pSV-TSG-6 (FIG. 9A) and pMAM-TSG-6 (FIG. 9B).

[0051]FIGS. 10A and 10B show Northern blot analysis of the expression ofTSG-6 mRNA in various stable transfectants. FIG. 10A shows blots ofcells transfected with pSV-TSG-6. FIG. 10B shows blots of cellstransfected with pMAMneo-TSG-6.

[0052]FIGS. 11A and 11B show Northern blot analysis of TNF induction ofTSG-6 mRNA in various cell lines. TNF (20 ng/ml) was added to confluentcells. After 4 hr, total RNA was extracted and subjected to Northernblot analysis. “CTL” (control) indicates no TNF treatment; “TNF”indicates 4-hour TNF treatment. The following cells were examined: FS-4:normal human diploid foreskin fibroblasts; GM-637: SV40-transformeddiploid fibroblast cell line; U937: human macrophage-like cell line fromhistiocytic lymphoma; A673: human rhabdomyosarcoma cell line; HUVEC:human umbilical vein endothelial cells; A549: human lung carcinoma cellline; Colo205: human colon aclenocarcinoma cell line; HT29: human colonadenocarcinorna cell line; MEL: SK-MEL-19, cutaneous malignant melanomacell line.

[0053]FIGS. 12A and 12B show a Northern blot analysis of TNF inductionof TSG-6 mRNA in fibroblasts and transformed fibroblast lines. TNF (20ng/ml) was added to confluent cells. Total RNA was extracted andsubjected to Northern blot analysis. FS-48 and FS-49 are normal humandiploid foreskin fibroblasts from different donors. WI-38 is a normalhuman diploid fetal lung fibroblast line. WI-38 VA13 is SV40-transformedWI-38 cell line. FS-4(SV1), FS-4(SV2) and FS-4(SV3) are FS-4 cellsimmortalized by lipofection with a pSV3-neo plasmid containing DNAencoding the SV40 large T antigen.

[0054]FIGS. 13A and 13B show a Western blot of concentrated supernatantsof serum-free cultures of FS-4 cells or transfected GM-637 cells. InFIG. 13A, bands were developed using anti-TSG-6 antibody purified byimmunoaffinity chromatography. In FIG. 13B, bands were developed withsimilarly purified pre-immune serum from the same rabbit. Lane 1:prestained molecular weight standards; Lane 2: supernatant of GM-637cells transfected with pRSVneo (GN4); Lane 3: supernatant of GM-637cells transfected with TSG-6 cDNA (GSV-L5); Lane 4: supernatant ofuntreated FS-4 cells; Lane 5: supernatant of FS-4 cells after 24 hrinduction with TNF (20 ng/ml); Lane 6: biotinylated molecular weightstandards.

[0055]FIG. 14 is a Western blot pattern showing that TSG-6 protein isdetectable in culture supernatants of GSV-15 cells, but not in celllysates. Supernatants of serum-free cultures of TSG-6 cDNA-transfectedGM-637 cells (GSV-L5 cells) and control-transfected GSV-neo cells wereconcentrated about 100-fold. To prepare lysates, cells were directlylysed in SDS-PAGE sample buffer. The samples were then subjected toWestern blot analysis using affinity-purified anti-TSG-6 antibody. Lane1: prestained molecular weight standards; lane 2: concentratedsupernatant of GSV-neo cells; lane 3: concentrated supernatant of GSV-L5cells; lane 4: lysate of GSV-neo cells; lane 5: lysate of GSV-L5 cells.

[0056]FIG. 15 is a Western blot pattern showing the binding of TSG-6protein to hyaluronic acid (HA) coupled to Sepharose. The concentratedsupernatant of GSV-L5 cells (serum-free) was incubated either withcontrol Sepharose (DEC-activated, acetic acid-blocked) (lanes 1, 2) orHA-Sepharose (lanes 3, 4) in a batch procedure. The supernatants (lanes1, 3) as well as the eluates (lanes 2, 4) were analyzed by Western blotwith anti-TSG-6 antibody. Lane 1: supernatant after absorption oncontrol Sepharose; lane 2: eluate from the control Sepharose; lane 3:supernatant after absorption on HA-Sepharose; lane 4: eluate fromHA-Sepharose.

[0057]FIG. 16 shows a Western blot analysis of TSG-6 protein eluted froma hyaluronic acid (HA)-Sepharose column. Unconcentrated supernatants ofGSV-L5 cells cultured in medium with 10% fetal calf serum was absorbedto a column of HA-Sepharose and eluted with Tris-HCl, pH 8.5, high saltbuffer. The eluate was analyzed by Western blot with affinity-purifiedanti-TSG-6 antibody.

[0058]FIG. 17 depicts a hypothetical model for the involvement of TSG-6in the release of proteoglycan in cartilage chondrocytes.

[0059]FIG. 18 shows that TSG-6 forms a stable complex with a serumprotein. Fetal bovine serum (lanes 2, 3), serum-free supernatants ofhuman HepG2 cells (lanes 4, 5), or mouse serum (lanes 6, 7) wasincubated in the absence (lanes 2, 4, 6) or presence (lanes 3, 5, 7) ofrecombinant human TSG-6 for 1 hr. at 37° C. All samples were thensubjected to Western blot analysis with rabbit antiserum to TSG-6. Lane1 is a TSG-6 control. The lower (29 kDa) TSG-6 band representsnonglycosylated protein present in variable amounts in preparations ofTSG-6 protein form insect cells infected with recombinant Baculovirus.

[0060]FIG. 19 shows immunoprecipitation of a ³⁵S-labeled 120-kDa proteincomplex by anti-TSG-6 antiserum. ³⁵S-labeled HepG2 culture supernatantwas preincubated for 1 hr. at 37° C. with (lanes 2, 3) or without(lane 1) unlabeled purified recombinant TSG-6 protein and precipitatedwith rabbit anti-TSG-6 immune serum (lanes 1, 2) or preimmune serum(lane 3) from the same rabbit. The samples were then analyzed bySDS-PAGE in a 10% PAA gel and fluorography.

[0061]FIG. 20 shows changes in the band pattern of a fraction of humanserum proteins after incubation with purified TSG-6 protein. A partiallypurified preparation of TSG-6 binding protein from human serum (afterfractioned ammonium sulfate precipitation, Affini-filter chromatography,and FPLC on MonoQ) was incubated in the absence (lane 2) or presence(lane 3) of purified TSG-6 protein at 37° C. for 1 hr. Lane 1 is a TSG-6control. The samples were separated by SDS-PAGE in a 10% PAA gel underreducing conditions and silver-stained.

[0062]FIG. 21 shows time course and temperature dependence of theformation of the TSG-6/IαI complex. Purified recombinant TSG-6 protein(lane 1) and IαI purified from human serum (lane 2) were mixed andincubated for 2 min. (lane 3), 5 min. (lane 4), 10 min. (lane 5), 15min. (lane 6), 30 min. (lane 7), or for 60 min. (lane 8) at 37° C. orfor 60 min. at 0° C. (lane 9). The reaction mixtures were then separatedby SDS-PAGE and subjected to Western blot analysis with rabbitanti-TSG-6 serum.

[0063]FIG. 22 shows binding of antibodies to I αI or TSG-6 proteinrecognize a 120-kDa complex formed after incubation of purified IαI andTSG-6 protein. Purified IαI was incubated in the absence (lanes 1, 4) orpresence (lanes 2, 5) or purified TSG-6 protein at 37° C. for 10 min.Lanes 3 and 6 contain purified TSG-6 protein without IαI. All sampleswere separated by SDS-PAGE on 8% PAA under reducing conditions. ForWestern blot analysis, lanes 1-3 were developed with anti-IαI, and lanes4-6 were developed with anti-TSG-6 antibody.

[0064]FIG. 23 shows treatment of the TSG-6/IαI complex with 8 M urea.100 μL of a TN-5 B insect cell culture supernatant containing TSG-6protein was incubated with 100 μL of a 1:50 dilution of human serum for1 hr. at 37° C. Thereafter, one 100-μL aliquot was mixed with 2 mL of 8M urea and concentrated in a Centricon-10 unit to 100 μL (lane 2); theother aliquot was left untreated (lane 1). The reaction mixtures werethen separated by SDS-PAGE and subjected to Western blot analysis withanti-TSG-6 antibody.

[0065]FIG. 24 shows treatment of the TSG-6/IαI complex with chondroitinsulfate ABC lyase or hyaluronidase. Partially purified IαI was incubatedin the absence (lane 1) or presence of purified TSG-6 protein (lanes3-5) at 37° C. for 1 hr. The sample containing both IαI and TSG-6protein was divided into aliquots and further incubated without enzyme(lane 3), with 800 milliunits of chondroitin sulfate ABC lyase (lane 4),or with 1.6 units of hyaluronidase (lane 5). Lane 2 is a TSG-6 controlwithout IαI. The reaction mixtures were separated by SDS-PAGE andanalyzed by Western blotting with anti-TSG-6 antibody.

[0066]FIG. 25 shows TSG-6/IαI complex formation by IαI and TSG-6proteins with chondroitin sulfate ABC lyase. Four micrograms of purifiedIαI and 3 μg of purified TSG-6 protein were incubated separately for 16hrs. at 37° C. in the absence or presence of 30 milliunits ofchondroitin sulfate ABC lyase. Thereafter, control IαI was mixed withcontrol TSG-6 protein (lane 1), chonroitinase-pretreated IαI was mixedwith control TSG-6 (lane 2), and control IαI was reaction mixtured withchondroitinase-pretreated TSG-6 protein (lane 3). The reaction mixtureswere incubated for 1 hr. at 37° C. before SDS-PAGE and Western blotanalysis with anti-TSG-6 antibody.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0067] A number of genes activated in human FS-4 fibroblasts by tumornecrosis factor (TNF) were termed by the present inventors“TNF-stimulated genes” (abbreviated TSG). It should be appreciated thatsuch genes, and the proteins and glycoproteins they encode, are inducedby cytokines more generally, including TNF, IL-1, and, in some case,interferons. The proteins, functional derivatives, such as peptidefragments, and antibodies to the proteins are useful in a number ofmethods of importance to the diagnosis and treatment of diseases andconditions in which the activity, or inactivity, of such cytokines isassociated with the pathophysiology. Such diseases include chronicinflammation, such as rheumatoid arthritis, cancer, and infections, inparticular with gram-negative bacteria.

[0068] Inflammatory conditions also detectable or treatable with aTSG-protein antibody, binding molecule or inhibiting molecule of thepresent invention can also include, but is not limited to, thefollowing, which can include TNF related pathologies:

[0069] (A) acute and chronic immune and autoimmune pathologies, such assystemic lupus erythematosus (SLE), rheumatoid arthritis, thyroidosis,graft versus host disease, scieroderma, diabetes mellitus, Graves'disease, and the like;

[0070] (B) infections, including, but not limited to, sepsis syndrome,cachexia, circulatory collapse and shock resulting from acute or chronicbacterial infection, acute and chronic parasitic and/or infectiousdiseases, bacterial, viral or fungal, such as HIV, AIDS (includingsymptoms of cachexia, autoimmune disorders, AIDS dementia complex andinfections);

[0071] (C) inflammatory diseases, such as chronic inflammatorypathologies and vascular inflammatory pathologies, including chronicinflammatory pathologies such as sarcoidosis, chronic inflammatory boweldisease, ulcerative colitis, and Crohn's pathology and vascularinflammatory pathologies, such as, but not limited to, disseminatedintravascular coagulation, atherosclerosis, and Kawasaki's pathology;

[0072] (D) neurodegenerative diseases, including, but are not limitedto,

[0073] demyelinating diseases, such as multiple sclerosis and acutetransverse myelitis;

[0074] extrapyramidal and cerebellar disorders such as lesions of thecorticospinal system;

[0075] disorders of the basal ganglia or cerebellar disorders;

[0076] hyperkinetic movement disorders such as Huntington's Chorea andsenile chorea;

[0077] drug-induced movement disorders, such as those induced by drugswhich block CNS dopamine receptors;

[0078] hypokinetic movement disorders, such as Parkinson's disease;

[0079] Progressive supranucleo palsy;

[0080] Cerebellar and Spinocerebellar Disorders, such as astructurallesions of the cerebellum; spinocerebellar degenerations (spinal ataxia,Friedreich's ataxia, cerebellar cortical degenerations, multiple systemsdegenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph);and systemic disorders (Refsum's disease, abetalipoprotemia, ataxia,telangiectasia, and mitochondrial multi-system disorder);

[0081] demyelinating core disorders, such as multiple sclerosis, acutetransverse myelitis;

[0082] disorders of the motor unit, such as neurogenic muscularatrophies (anterior horn cell degeneration, such as amyotrophic lateralsclerosis, infantile spinal muscular atrophy and juvenile spinalmuscular atrophy); Alzheimer's disease; Down's Syndrome in middle age;Diffuse Lewy body disease; Senile Dementia of Lewy body type;Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakobdisease; Subacute sclerosing panencephalitis, Hallerrorden-Spatzdisease; and Dementia pugilistica, or any subset thereof;

[0083] (E) malignant pathologies involving TNF-secreting tumors or othermalignancies involving TNF, such as, but not limited to leukemias(acute, chronic myelocytic, chronic lymphocytic and/or myelodyspasticsyndrome); lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such asmalignant lymphomas (Burkitt's lymphoma or Mycosis fungoides)); and

[0084] (F) alcohol-induced hepatitis.

[0085] See, e.g., Berkow et al, eds., The Merck Manual, 16th edition,chapter 11, pp 1380-1529, Merck and Co., Rahway, N.J., 1992, whichreference, and references cited therein, are entirely incorporatedherein by reference.

[0086] The present invention is directed to one of these genes and itsprotein product, both termed TSG-6. The present invention provides TSG-6DNA, mRNA and protein in substantially pure form, functional derivativesof the protein such as peptide fragments, antibodies specific for theprotein, methods of producing the DNA, mRNA and protein, methods ofusing these molecules in diagnosis, therapy, and study of theabove-mentioned disease states.

[0087] By “substantially pure” is meant any protein or peptide of thepresent invention, or any DNA or mRNA sequence encoding any such proteinor peptide, which is essentially free of other proteins, DNA sequencesor mRNA sequences, respectively, or of other contaminants with which itmight normally be found in nature, and, as such, exists in a form notfound in nature.

[0088] “Substantially free of other proteins” indicates that the proteinhas been purified away from at least 90 percent (on a weight basis), andfrom even at least 99 percent, if desired, of other proteins andglycoproteins with which it is natively associated, and is thereforesubstantially free of them. That can be achieved by subjecting thecells, tissue or fluids expressing or containing the TSG-6 protein toprotein purification techniques such as immunoabsorbent columns bearingantibodies, such as monoclonal antibodies (mAb) reactive against theprotein. Because of the fact that TSG-6 binds to hyaluronic acid, theTSG-6 protein or glycoprotein may be purified using an affinity columnto which hyaluronic acid is bound. Alternatively, the purification canbe achieved by a combination of standard methods, such as ammoniumsulfate precipitation, molecular sieve chromatography, and ion exchangechromatography.

[0089] The methods of the present invention are used to identify normalor mutant TSG-6 genes or measure the presence or amount of TSG-6 proteinassociated with a cell or tissue, or secreted by a cell; such methodscan be used to identify susceptibility to, or presence of (a)inflammatory conditions, in particular proteoglycan breakdown such asthat associated with rheumatoid arthritis, (b) sepsis followinggram-negative bacterial infections, and (c) disorders associated withleukocyte adhesion.

[0090] An amino acid or nucleic acid sequence of a TSG-6 polypeptide ofthe present invention is said to “substantially correspond” to anotheramino acid or nucleic acid sequence, respectively, if the sequence ofamino acids or nucleic acid in both molecules provides polypeptideshaving biological activity that is substantially similar, qualitativelyor quantitatively, to the corresponding fragment of at least onehyaluron binding domain, an inter-α-inhibitor binding domain, a TNFbinding domain, or which may be synergistic when two or more of thesedomains, consensus sequences or homologs thereof are present.

[0091] Additionally or alternatively, such “substantially corresponding”sequences of TSG-6 polypeptides include conservative amino acid ornucleotide substitutions, or degenerate nucleotide codon substitutionswherein individual amino acid or nucleotide substitutions are well knownin the art.

[0092] Alternatively or additionally, substantially corresponding refersto TSG-6 polypeptides having amino acid sequences having at least 80%homology or identity to an amino acid sequence of SEQ ID NO:1 or 2, suchas 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99 or 100% homology or identity, e.g., using known modelingalgorithms, such as, but not limited to, ECEPP, INSIGHT, DISCOVER,CHEM-DRAW, AMBER, FRODO and CHEM-X. Such algorithms compare bindingdomains between related TSG-6 polypeptides, and alternative consensuspolypeptide fragments are thus determined.

[0093] Accordingly, TSG-6 polypeptides of the present invention, ornucleic acid encoding therefor, include a finite set of substantiallycorresponding sequences as substitution peptides or polynucleotideswhich can be routinely obtained by one of ordinary skill in the art,without undue experimentation, based on the teachings and guidancepresented herein. For a detailed description of protein chemistry andstructure, see Schulz, G. E. et al, Principles of Protein Structure,Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins:Structure and Molecular Properties, W. H. Freeman & Co., San Francisco,1983, which are hereby incorporated by reference. For a presentation ofnucleotide sequence substitutions, such as codon preferences, seeAusubel et al, eds., Current Protocols in Molecular Biology, WileyIntersciences, NY (1987-1995) at □□A.1.1-A.1.24, and Sambrook et al, In:Molecular Cloning: A Laboratory Manual, second edition, Cold SpringHarbor Press, NY (1989), at Appendices C and D.

[0094] Conservative substitutions of a TSG-6 polypeptide of the presentinvention includes a variant wherein at least one amino acid residue inthe polypeptide has been conservatively replaced by a different aminoacid. Such substitutions preferably are made in accordance with thefollowing list as presented in Table IA which substitutions may bedetermined by routine experimentation to provide modified structural andfunctional properties of a synthesized polypeptide molecule, whilemaintaining the receptor binding, inhibiting or mimicking biologicalactivity, as determined by TSG-6 binding receptor activity assays. TABLEIA Original Exemplary Residue Substitution Ala Gly; Ser Arg Lys Asn Gln;His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala; Pro His Asn; Gln Ile Leu;Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Tyr; Ile Phe Met; Leu; TyrSer Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0095] Alternatively, another group of substitutions of TSG-6polypeptides of the present invention are those in which at least oneamino acid residue in the protein molecule has been removed and adifferent residue inserted in its place according to the following TableIB. The types of substitutions which may be made in the protein orpeptide molecule of the present invention may be based on analysis ofthe frequencies of amino acid changes between a homologous protein ofdifferent species, such as those presented in Table 1-2 of Schulz et al,supra and FIGS. 3-9 of Creighton, supra. Based on such an analysis,alternative conservative substitutions are defined herein as exchangeswithin one of the following five groups: TABLE IB 1. Small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polarnegatively charged residues and their amides: Asp, Asn, Glu, Gln; 3.Polar, positively charged residues: His, Arg, Lys; 4. Large aliphaticnonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromaticresidues: Phe, Tyr, Trp.

[0096] The three amino acid residues in parentheses above have specialroles in protein architecture. Gly is the only residue lacking any sidechain and thus imparts flexibility to the chain. This however tends topromote the formation of secondary structure other than α-helical. Pro,because of its unusual geometry, tightly constrains the chain. Itgenerally tends to promote β-turn-like structures, although in somecases Cys can be capable of participating in disulfide bond formationwhich is important in protein folding. Note the Schulz et al would mergeGroups 1 and 2, above. Note also that Tyr, because of its hydrogenbonding potential, has significant kinship with Ser, and Thr, etc.

[0097] Conservative amino acid substitutions according to the presentinvention, e.g., as presented above, are known in the art and would beexpected to maintain biological and structural properties of thepolypeptide after amino acid substitution. Most deletions andinsertions, and substitutions according to the present invention arethose which do not produce radical changes in the characteristics of theprotein or peptide molecule. “Characteristics” is defined in anon-inclusive manner to define both changes in secondary structure, e.g.α-helix or β-sheet, as well as changes in physiological activity, e.g.,in receptor binding assays.

[0098] However, when the exact effect of the substitution, deletion, orinsertion is to be confirmed, one skilled in the art will appreciatethat the effect of the substitution or substitutions will be evaluatedby routine screening assays, either immunoassays or bioassays to confirmbiological activity, such as receptor binding or modulation of ligandbinding to the corresponding TSG-6 receptor. See, e.g., Maranges et al,eds. A substituted polypeptide typically is made by site-specificmutagenesis of the peptide molecule-encoding nucleic acid, expression ofthe mutant nucleic acid in recombinant cell culture, and, optionally,purification from the cell culture, for example, by immunoaffinitychromatography using a specific antibody on a chemically derivatizedcolumn or immobilized membranes or hollow fibers (to absorb the mutantby binding to at least one epitope).

[0099] In one embodiment, the invention is directed to a naturallyoccurring TSG-6 protein or glycoprotein substantially free fromimpurities of human origin with which it is natively associated. Inanother embodiment, the invention is directed to a recombinant TSG-6encoded protein or glycoprotein.

[0100] It will be understood that the TSG-6 protein of the presentinvention can be purified biochemically or physicochemically from avariety of cell or tissue sources. For preparation of naturallyoccurring TSG-6 protein, connective tissue cells such as humanfibroblasts are preferred. Alternatively, methods are well known for thesynthesis of polypeptides of desired sequence on solid phase supportsand their subsequent separation from the support.

[0101] Because the TSG-6 gene can be isolated or synthesized, the TSG-6polypeptide, or a functional derivative thereof, can be synthesizedsubstantially free of other proteins or glycoproteins of mammalianorigin in a prokaryotic organism or in a non-mammalian eukaryoticorganism, if desired. As intended by the present invention, a TSG-6protein or glycoprotein molecule produced by recombinant means inmammalian cells, such as transfected GM-637 cells, for example, iseither a naturally occurring protein sequence or a functional derivativethereof. Where a naturally occurring protein or glycoprotein is producedby recombinant means, it is provided substantially free of the otherproteins and glycoproteins with which it is natively associated.

[0102] A preferred use of this invention is the production by chemicalsynthesis or recombinant DNA technology of fragments of the TSG-6molecule, which still retain biological activity such as binding toantibodies, binding to hyaluronic acid, and the like. Among theadvantages of shorter peptides for some of the methods of the presentinvention are (1) greater stability and diffusibility, and (2) lessimmunogenicity. As discussed herein, the TSG-6 proteins or peptides ofthe present invention may be further modified for purposes of drugdesign, such as, for example, to reduce immunogenicity, to promotesolubility or enhance delivery, or to prevent clearance or degradation.

[0103] Also included within the scope of the present invention aresoluble forms of the TSG-6 protein, and functional derivatives of theTSG-6 protein having similar bioactivity for all the uses describedherein. Also intended are all active forms of TSG-6 derived from theTSG-6 transcript, and all muteins with TSG-6 activity.

[0104] By “functional derivative” is meant a “fragment,” “variant,”“analog,” or “chemical derivative” of the TSG-6 protein. A functionalderivative retains at least a portion of the function of the TSG-6protein which permits its utility in accordance with the presentinvention.

[0105] A “fragment” of the TSG-6 protein is any subset of the molecule,that is, a shorter peptide.

[0106] A “variant” of the TSG-6 refers to a molecule sub-stantiallysimilar to either the entire peptide or a fragment thereof. Variantpeptides may be conveniently prepared by direct chemical synthesis ofthe variant peptide, using methods well-known in the art.

[0107] Alternatively, amino acid sequence variants of the peptide can beprepared by mutations in the DNA which encodes the synthesized peptide.Such variants include, for example, deletions from, or insertions orsubstitutions of, residues within the amino acid sequence. Anycombination of deletion, insertion, and substitution may also be made toarrive at the final construct, provided that the final constructpossesses the desired activity. Obviously, the mutations that will bemade in the DNA encoding the variant peptide must not alter the readingframe and preferably will not create complementary regions that couldproduce secondary mRNA structure (see European Patent Publication No. EP75,444).

[0108] At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis (as exemplified by Adelman et al, DNA 2:183(1983)) of nucleotides in the DNA encoding the peptide molecule, therebyproducing DNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture. The variants typically exhibit the samequalitative biological activity as the nonvariant peptide.

[0109] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al, Proc.Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is then annealed withthe single-stranded protein-sequence-containing vector, and subjected toDNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment,to complete the synthesis of the mutation-bearing strand. Thus, amutated sequence in the second strand bears the desired mutation. Thisheteroduplex vector is then used to transform appropriate cells andclones are selected that include recombinant vectors bearing the mutatedsequence arrangement. The mutated protein region may be removed andplaced in an appropriate vector for protein production, generally anexpression vector of the type that may be employed for transformation ofan appropriate host.

[0110] Alternatively, the DNA encoding a normal or variant TSG-6 proteincan be altered by homologous recombination, a technique developed withinthe past few years for targeting genes to induce or correct mutations intranscriptionally active genes (Kucherlapati, Prog. in Nucl. Acid Res.and Mol. Biol. 36:301 (1989)). The technique of homologous recombinationwas developed as a method for introduction of specific mutations intospecific regions of the mammalian genome (Thomas et al, Cell,44:419-428, 1986; Thomas and Capecchi, Cell 51:503-512 (1987);Doetschman et al, Proc. Natl. Acad. Sci. USA 85:8583-8587 (1988)) or tocorrect specific mutations within defective genes (Doetschman et al,Nature 330:576-578 (1987)). The above references to homologousrecombination are hereby incorporated by reference.

[0111] An example of a terminal insertion includes a fusion of a signalsequence, whether heterologous or homologous to the host cell, to theN-terminus of the peptide molecule to facilitate the secretion of maturepeptide molecule from recombinant hosts.

[0112] Another group of variants are those in which at least one aminoacid residue in the peptide molecule, and preferably, only one, has beenremoved and a different residue inserted in its place. Suchsubstitutions preferably are made in accordance with the following listwhen it is desired to modulate finely the characteristics of a peptidemolecule. Original Exemplary Original Exemplary Residue SubstitutionResidue Substitution Ala Gly; Ser Leu Ile; Val Arg Lys Lys Arg; Gln; GluAsn Gln; His Met Leu; Tyr; Ile Asp Glu Phe Met; Leu; Tyr Cys Ser Ser ThrGln Asn Thr Ser Glu Asp Trp Tyr Gly Ala; Pro Tyr Trp; Phe His Asn; GlnVal Ile; Leu Ile Leu; Val

[0113] Substantial changes in functional or immunological properties aremade by selecting substitutions that are less conservative than those inthe above list, that is, by selecting residues that differ moresignificantly in their effect on maintaining (a) the structure of thepeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Thesubstitutions that in general are expected to produce substantialchanges are those in which (a) glycine and/or proline is substituted byanother amino acid or is deleted or inserted; (b) a hydrophilic residue,e.g., seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) acysteine residue is substituted for (or by) any other residue; (d) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistidyl, is substituted for (or by) a residue having an electronegativecharge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky sidechain, e.g., phenylalanine, is substituted for (or by) one not havingsuch a side chain, e.g., glycine.

[0114] Most deletions and insertions, and substitutions in particular,are not expected to produce radical changes in the characteristics ofthe peptide molecule. However, when it is difficult to predict the exacteffect of the substitution, deletion, or insertion in advance of doingso, one skilled in the art will appreciate that the effect will beevaluated by routine screening assays. For example, a TSG-6 varianttypically is made by site-specific mutagenesis or homologousrecombination of the TSG-6-encoding nucleic acid, expression of thevariant nucleic acid in recombinant cell culture, and, optionally,purification from the cell culture, for example, by immunoaffinityadsorption on an antibody containing column.

[0115] An “analog” of the TSG-6 protein refers to a non-natural moleculesubstantially similar to either the entire molecule or a fragmentthereof.

[0116] A “chemical derivative” of the TSG-6 protein contains additionalchemical moieties not normally a part of the protein. Covalentmodifications of the peptide are included within the scope of thisinvention. Such modifications may be introduced into the molecule byreacting targeted amino acid residues of the peptide with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues.

[0117] Cysteinyl residues most commonly are reacted withalpha-haloacetates (and corresponding amines), such as 2-chloroaceticacid or chloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0118] Histidyl residues are derivatized by reaction withdiethylprocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Parabromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0.

[0119] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing alpha-amino-containing residuesinclude imidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0120] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0121] The specific modification of tyrosyl residues per se has beenstudied extensively, with particular interest in introducing spectrallabels into tyrosyl residues by reaction with aromatic diazoniumcompounds or tetranitromethane. Most commonly, N-acetylimidizol andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

[0122] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R′-N-C-N-R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

[0123] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

[0124] Derivatization with bifunctional agents is useful forcross-linking the peptide to a water-insoluble support matrix or toother macromolecular carriers. Commonly used cross-linking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succin-imidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

[0125] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

[0126] Such derivatized moieties may improve the solubility, absorption,biological half life, and the like. The moieties may alternativelyeliminate or attenuate any undesirable side effect of the protein andthe like. Moieties capable of mediating such effects are disclosed, forexample, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

[0127] Standard reference works setting forth the general principles ofrecombinant DNA technology include Watson, J. D. et al, MolecularBiology of the Gene, Volumes I and II, The Benjamin/Cummings PublishingCompany, Inc., publisher, Menlo Park, Calif. (1987); Darnell, J. E. etal, Molecular Cell Biology, Scientific American Books, Inc., publisher,New York, N.Y. (1986); Lewin, B. M., Genes II, John Wiley & Sons,publishers, New York, N.Y. (1985); Old, R. W., et al, Principles of GeneManipulation: An Introduction to Genetic Engineering, 2d edition,University of California Press, publisher, Berkeley, Calif. (1981); andSambrook, J. et al, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989) and Ausubel et al,supra. These references are hereby incorporated by reference.

[0128] By “cloning” is meant the use of in vitro recombinationtechniques to insert a particular gene or other DNA sequence into avector molecule. In order to successfully clone a desired gene, it isnecessary to employ methods for generating DNA fragments, for joiningthe fragments to vector molecules, for introducing the composite DNAmolecule into a host cell in which it can replicate, and for selectingthe clone having the target gene from amongst the recipient host cells.

[0129] By “cDNA” is meant complementary or copy DNA produced from an RNAtemplate by the action of RNA-dependent DNA polymerase (reversetranscriptase). Thus a “cDNA clone” means a duplex DNA sequencecomplementary to an RNA molecule of interest, carried in a cloningvector.

[0130] By “cDNA library” is meant a collection of recombinant DNAmolecules containing cDNA inserts which together comprise the entireexpressible genome of an organism. Such a cDNA library may be preparedby methods known to those of skill, and described, for example, inSambrook et al, supra, and Ausubel et al, supra. Generally, RNA is firstisolated from the cells of an organism from whose genome it is desiredto clone a particular gene. Preferred for the purposes of the presentinvention are mammalian, most preferably, human, cell lines.

[0131] Oligonucleotides representing a portion of the TSG-6 sequence areuseful for screening for the presence of homologous genes and for thecloning of such genes. Techniques for synthesizing such oligonucleotidesare disclosed by, for example, Wu, R., et al, Prog. Nucl. Acid. Res.Molec. Biol. 21:101-141 (1978).

[0132] Because the genetic code is degenerate, more than one codon maybe used to encode a particular amino acid (Watson, J. D., In: MolecularBiology of the Gene, 4th Ed., Benjamin/Cummings Publishing Co., Inc.,Menlo Park, Calif. (1987)). Using the genetic code, one or moredifferent oligonucleotides can be identified, each of which would becapable of encoding the amino acid. The probability that a particularoligonucleotide will, in fact, constitute the actual XXX-encodingsequence can be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic cells.Such “codon usage rules” are disclosed by Lathe, R., et al, J. Molec.Biol. 183:1-12 (1985). Using the “codon usage rules” of Lathe, a singleoligonucleotide, or a set of oligonucleotides, that contains atheoretical “most probable” nucleotide sequence capable of encoding theTSG-6 sequences is identified.

[0133] Although occasionally an amino acid sequence may be encoded byonly a single oligonucleotide, frequently the amino acid sequence may beencoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of this set contain oligonucleotides whichare capable of encoding the TSG-6 peptide fragment and, thus,potentially contain the same oligonucleotide sequence as the gene whichencodes the peptide fragment, only one member of the set contains thenucleotide sequence that is identical to the nucleotide sequence of thegene. Because this member is present within the set, and is capable ofhybridizing to DNA even in the presence of the other members of the set,it is possible to employ the unfractionated set of oligonucleotides inthe same manner in which one would employ a single oligonucleotide toclone the gene that encodes the protein.

[0134] The oligonucleotide, or set of oligonucleotides, containing thetheoretical “most probable” sequence capable of encoding the TSG-6fragment is used to identify the sequence of a complementaryoligonucleotide or set of oligonucleotides which is capable ofhybridizing to the “most probable” sequence, or set of sequences. Anoligonucleotide containing such a complementary sequence can be employedas a probe to identify and isolate the TSG-6 gene (Sambrook et al,supra).

[0135] A suitable oligonucleotide, or set of oligonucleotides, which iscapable of encoding a fragment of the TSG-6 gene (or which iscomplementary to such an oligonucleotide, or set of oligonucleotides) isidentified (using the above-described procedure), synthesized, andhybridized by means well known in the art, against a DNA or, morepreferably, a cDNA preparation derived from cells which are capable ofexpressing the TSG-6 gene, such as TNF-treated FS-4 cells.

[0136] Single stranded oligonucleotide molecules complementary to the“most probable” TSG-6 protein coding sequences can be synthesized usingprocedures which are well known to those of ordinary skill in the art(Belagaje, R., et al, J. Biol. Chem. 254:5765-5780 (1979); Maniatis, T.,et al, In: Molecular Mechanisms in the Control of Gene Expression,Nierlich, D. P., et al, Eds., Acad. Press, NY (1976); Wu, R., et al,Prog. Nucl. Acid Res. Molec. Biol. 21:101-141 (1978); Khorana, R. G.,Science 203:614-625 (1979)). Additionally, DNA synthesis may be achievedthrough the use of automated synthesizers. Techniques of nucleic acidhybridization are disclosed by Sambrook et al (supra), and by Haymes, B.D., et al (In: Nucleic Acid Hybridization, A Practical Approach, IRLPress, Washington, D.C. (1985)), which references are hereinincorporated by reference. Techniques such as, or similar to, thosedescribed above have successfully enabled the cloning of genes for humanaldehyde dehydrogenases (Hsu, L. C., et al, Proc. Natl. Acad. Sci. USA82:3771-3775 (1985)), fibronectin (Suzuki, S., et al, Eur. Mol. Biol.Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter,P., et al, Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-typeplasminogen activator (Pennica, D., et al, Nature 301:214-221 (1983))and human term placental alkaline phosphatase complementary DNA (Kam,W., et al, Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

[0137] In an alternative way of cloning the TSG-6 gene, a library ofexpression vectors is prepared by cloning DNA or, more preferably, cDNA(from a cell capable of expressing TSG-6, such as a TNF-treated FS-4cell) into an expression vector. The library is then screened formembers capable of expressing a protein which binds to anti-TSG-6antibody, and which has a nucleotide sequence that is capable ofencoding polypeptides that have the same amino acid sequence as TSG-6proteins or peptides, or fragments thereof. In this embodiment, DNA, ormore preferably cDNA, is extracted and purified from a cell which iscapable of expressing TSG-6 protein. The purified cDNA is fragmentized(by shearing, endonuclease digestion, etc.) to produce a pool of DNA orcDNA fragments. DNA or cDNA fragments from this pool are then clonedinto an expression vector in order to produce a genomic library ofexpression vectors whose members each contain a unique cloned DNA orcDNA fragment.

[0138] By “vector” is meant a DNA molecule, derived from a plasmid orbacteriophage, into which fragments of DNA may be inserted or cloned. Avector will contain one or more unique restriction sites, and may becapable of autonomous replication in a defined host or vehicle organismsuch that the cloned sequence is reproducible.

[0139] An “expression vector” is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA (or cDNA) molecule which has been clonedinto the vector and of thereby producing a polypeptide or protein.Expression of the cloned sequences occurs when the expression vector isintroduced into an appropriate host cell. If a prokaryotic expressionvector is employed, then the appropriate host cell would be anyprokaryotic cell capable of expressing the cloned sequences. Similarly,if a eukaryotic expression vector is employed, then the appropriate hostcell would be any eukaryotic cell capable of expressing the clonedsequences. Importantly, since eukaryotic DNA may contain interveningsequences, and since such sequences cannot be correctly processed inprokaryotic cells, it is preferable to employ cDNA from a cell which iscapable of expressing TSG-6 in order to produce a prokaryotic genomicexpression vector library. Procedures for preparing cDNA and forproducing a genomic library are disclosed by Sambrook et al (supra).

[0140] By “functional derivative” of a polynucleotide (DNA or RNA)molecule is meant a polynucleotide molecule encoding a “fragment” or“variant” of the TSG-6 protein. It can be a chemical derivative whichretains its functions such as the ability to hybridize with acomplementary polynucleotide molecule. Such a polynucleotide, oroligonucleotide, chemical derivative is useful as a molecular probe todetect TSG-6 sequences through nucleic acid hybridization assays.

[0141] A DNA sequence encoding the TSG-6 protein of the presentinvention, or its functional derivatives, may be recombined with vectorDNA in accordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed by Sambrook, J. et al, supra, and are well known in the art.

[0142] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression. The precise natureof the regulatory regions needed for gene expression may vary fromorganism to organism, but shall in general include a promoter regionwhich, in prokaryotes, contains both the promoter (which directs theinitiation of RNA transcription) as well as the DNA sequences which,when transcribed into RNA, will signal the initiation of proteinsynthesis. Such regions will normally include those 5′-non-codingsequences involved with initiation of transcription and translation,such as the TATA box, capping sequence, CAAT sequence, and the like.

[0143] If desired, the non-coding region 3′ to the gene sequence codingfor the protein may be obtained by the above-described methods. Thisregion may be retained for its transcriptional termination regulatorysequences, such as termination and polyadenylation. Thus, by retainingthe 3′-region naturally contiguous to the DNA sequence coding for theprotein, the transcriptional termination signals may be provided. Wherethe transcriptional termination signals are not satisfactorilyfunctional in the expression host cell, then a 3′ region functional inthe host cell may be substituted.

[0144] Two sequences of a nucleic acid molecule are said to be “operablylinked” when they are linked to each other in a manner which eitherpermits both sequences to be transcribed onto the same RNA transcript,or permits an RNA transcript, begun in one sequence to be extended intothe second sequence. Thus, two sequences, such as a promoter sequenceand any other “second” sequence of DNA or RNA are operably linked iftranscription commencing in the promoter sequence will produce an RNAtranscript of the operably linked second sequence. In order to be“operably linked” it is not necessary that two sequences be immediatelyadjacent to one another.

[0145] A promoter is a double-stranded DNA or RNA molecule which iscapable of binding RNA polymerase and promoting the transcription of an“operably linked” nucleic acid sequence. As used herein, a “promotersequence” is the sequence of the promoter which is found on that strandof the DNA or RNA which is transcribed by the RNA polymerase. A“promoter sequence complement” is a nucleic acid molecule whose sequenceis the complement of a “promoter sequence.” Hence, upon extension of aprimer DNA or RNA adjacent to a single-stranded “promoter sequencecomplement” or, of a “promoter sequence,” a double-stranded molecule iscreated which will contain a functional promoter, if that extensionproceeds towards the “promoter sequence” or the “promoter sequencecomplement.” This functional promoter will direct the transcription of anucleic acid molecule which is operably linked to that strand of thedouble-stranded molecule which contains the “promoter sequence” (and notthat strand of the molecule which contains the “promoter sequencecomplement”).

[0146] Certain RNA polymerases exhibit a high specificity for suchpromoters. The RNA polymerases of the bacteriophages T7, T3, and SP-6are especially well characterized, and exhibit high promoterspecificity. The promoter sequences which are specific for each of theseRNA polymerases also direct the polymerase to utilize (i.e. transcribe)only one strand of the two strands of a duplex DNA template. Theselection of which strand is transcribed is determined by theorientation of the promoter sequence. This selection determines thedirection of transcription since RNA is only polymerized enzymaticallyby the addition of a nucleotide 5′ phosphate to a 3′ hydroxyl terminus.The promoter sequences of the present invention may be eitherprokaryotic, eukaryotic or viral. Suitable promoters are repressible,or, more preferably, constitutive. Strong promoters are preferred.

[0147] The present invention encompasses the expression of the TSG-6protein (or a functional derivative thereof) in either prokaryotic oreukaryotic cells, although eukaryotic expression is preferred.

[0148] Preferred prokaryotic hosts include bacteria such as E. coli,Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The mostpreferred prokaryotic host is E. coli. Other enterobacteria such asSalmonella typhimurium or Serratia marcescens, and various Pseudomonasspecies may also be utilized. Under such conditions, the protein may notbe glycosylated. The procaryotic host must be compatible with thereplicon and control sequences in the expression plasmid.

[0149] The TSG-6 protein can be expressed in a prokaryotic cell (suchas, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.),either by itself, or as part of a fusion protein. For expression as afusion protein, it must be linked in the appropriate reading frame witha prokaryotic protein. Preferred fusion protein “partners” are the trpEprotein of E. coli or a bacteriophage protein, such as that of the MS2phage (see Examples, below). To express the TSG-6 protein (or afunctional derivative thereof) in a prokaryotic host, it is necessary tooperably link the TSG-6 encoding sequence to a functional prokaryoticpromoter. Examples of constitutive promoters include the int promoter ofbacteriophage lambda, the bla promoter of the β-lactamase gene ofpBR322, and the CAT promoter of the chloramphenicol acetyl transferasegene of pBR325, etc. Examples of inducible prokaryotic promoters includethe major right and left promoters of bacteriophage lambda (P_(L) andP_(R)), the trp, recA, lacZ, lacI, and gal promoters of E. coli, theα-amylase (Ulmanen, I., et al, J. Bacteriol. 162:176-182 (1985)) and thes-28-specific promoters of B. subtilis (Gilman, M. Z., et al, Gene32:11-20 (1984)), the promoters of the bacteriophages of Bacillus(Gryczan, T. J., In: The Molecular Biology of the Bacilli, AcademicPress, Inc., NY (1982)), and Streptomyces promoters (Ward, J. M., et al,Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewedby Glick, B. R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y.(Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet.18:415-442 (1984)). For the present invention, a most preferred promoteris the PL promoter of lambda; alternatively, the protein can beexpressed under control of a temperature-sensitive repressor of thelambda PL promoter (see Examples, below).

[0150] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream of the gene-encodingsequence. Such ribosome binding sites are disclosed, for example, byGold, L., et al (Ann. Rev. Microbiol. 35:365-404 (1981)).

[0151] Preferred hosts are eukaryotic hosts including yeast, insects,fungi, and mammalian cells either in vivo, or in tissue culture.Mammalian cells provide post-translational modifications to proteinmolecules including correct folding or glycosylation at correct sites.Mammalian cells which may be useful as hosts include cells of fibroblastorigin such as VERO or CHO, or cells of lymphoid origin, such as thehybridoma SP2/O-Ag14 or the murine myeloma P3-X63Ag8, and theirderivatives. A most preferred host is one that does not express theTSG-6 gene upon treatment with TNF, such as GM-637, a SV40-transformedhuman fibroblast cell line.

[0152] For a mammalian cell host, many possible vector systems areavailable for the expression of TSG-6. A wide variety of transcriptionaland translational regulatory sequences may be employed, depending uponthe nature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, Simian virus 40, or the like, where the regulatorysignals are associated with a particular gene which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the genes can be modulated. Ofinterest are regulatory signals which are temperature-sensitive so thatby varying the temperature, expression can be repressed or initiated, orare subject to chemical regulation, e.g., metabolite.

[0153] A yeast cell host provides substantial advantages in that it canalso carry out post-translational peptide modifications includingglycosylation. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number of plasmids whichcan be utilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene products andsecretes peptides bearing leader sequences (i.e., pre-peptides).

[0154] Any of a series of yeast gene expression systems incorporatingpromoter and termination elements from the actively expressed genescoding for glycolytic enzymes produced in large quantities when yeastare grown in mediums rich in glucose can be utilized. Known glycolyticgenes can also provide very efficient transcriptional control signals.For example, the promoter and terminator signals of the phosphoglyceratekinase gene can be utilized.

[0155] Production of TSG-6 or functional derivatives thereof in insectscan be achieved, for example, by infecting the insect host with abaculovirus engineered to express TSG-6 by methods known to those ofskill. Thus, in one embodiment, sequences encoding TSG-6 may be operablylinked to the regulatory regions of the viral polyhedrin protein (Jasny,Science 238: 1653 (1987)). Infected with the recombinant baculovirus,cultured insect cells, or the live insects themselves, can produce theTSG-6 protein in amounts as great as 20 to 50% of total proteinproduction. When live insects are to be used, caterpillars are presentlypreferred hosts for large scale TSG-6 production according to theinvention.

[0156] As discussed above, expression of the TSG-6 protein in eukaryotichosts requires the use of eukaryotic regulatory regions. Such regionswill, in general, include a promoter region sufficient to direct theinitiation of RNA synthesis. Preferred eukaryotic promoters include theSV40 early promoter (Benoist, C., et al, Nature (London) 290:304-310(1981)); the RSV promoter associated with an MMTV LTR region; promoterof the mouse metallothionein I gene (Hamer, D., et al, J. Mol. Appl.Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S.,Cell 31:355-365 (1982)); the yeast gal4 gene promoter (Johnston, S. A.,et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P. A.,et al, Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

[0157] As is widely known, translation of eukaryotic mRNA is initiatedat the codon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the TSG-6 protein (or a functionalderivative thereof) does not contain any intervening codons which arecapable of encoding a methionine (i.e., AUG). The presence of suchcodons results either in a formation of a fusion protein (if the AUGcodon is in the same reading frame as TSG-6 encoding DNA sequence) or aframe-shift mutation (if the AUG codon is not in the same reading frameas the TSG-6 encoding sequence).

[0158] The TSG-6 encoding sequence and an operably linked promoter maybe introduced into a recipient prokaryotic or eukaryotic cell either asa non-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the TSG-6 protein may occur through the transient expression of theintroduced sequence. Alternatively, permanent expression may occurthrough the integration of the introduced sequence into the hostchromosome.

[0159] In one embodiment, a vector is employed which is capable ofintegrating the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker may provide for prototropy to an auxotrophic host,biocide resistance, e.g., antibiotics, or heavy metals, such as copperor the like. The selectable marker gene can either be directly linked tothe DNA gene sequences to be expressed, or introduced into the same cellby co-transfection. Additional elements may also be needed for optimalsynthesis of single chain binding protein mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama, H., Mol. Cell. Biol. 3:28(1983).

[0160] In a preferred embodiment, the introduced sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include: the ease with whichrecipient cells that contain the vector may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species. Preferred prokaryotic vectors includeplasmids such as those capable of replication in E. coli (such as, forexample, pBR322, ColE1, pSC101, pACYC 184,

VX. Such plasmids are, for example, disclosed by Sambrook et al (supra).Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids aredisclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli,Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmidsinclude pIJ101 (Kendall, K. J., et al, J. Bacteriol. 169:4177-4183(1987)), and streptomyces bacteriophages such as φC31 (Chater, K. F., etal, In: Sixth International Symposium on Actinomycetales Biology,Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonasplasmids are reviewed by John, J. F., et al (Rev. Infect. Dis. 8:693-704(1986)), and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978)).

[0161] Preferred eukaryotic plasmids include BPV, vaccinia, SV40,2-micron circle, etc., or their derivatives. Such plasmids are wellknown in the art (Botstein, D., et al, Miami Wntr. Symp. 19:265-274(1982); Broach, J. R., In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982); Bollon, D. P., et al, J. Clin. Hematol. Oncol.10:39-48 (1980); Maniatis, T., In: Cell Biology: A ComprehensiveTreatise, Vol. 3, Gene Expression, Academic Press, NY, pp. 563-608(1980)).

[0162] Once the vector or DNA sequence containing the construct(s) hasbeen prepared for expression, the vector or DNA construct(s) may beintroduced into an appropriate host cell by any of a variety of suitablemeans, including such biochemical means as transformation, transfection,conjugation, protoplast fusion, calcium phosphate-precipitation, andapplication with lipid-based carriers and with polycations such asdiethylaminoethyl (DEAE) dextran, and such mechanical means aselectroporation, direct microinjection, and microprojectile bombardment(Johnston et al, Science 240:1538 (1988)), etc.

[0163] After the introduction of the vector, recipient cells are grownin a selective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the TSG-6 protein, or in the production of a fragment ofthis protein. This can take place in the transformed cells as such, orfollowing the induction of these cells to differentiate.

[0164] The expressed protein or fusion protein may be isolated andpurified in accordance with conventional conditions, such as extraction,precipitation, chromatography, affinity chromatography, electrophoresis,or the like. For example, the cells may be collected by centrifugation,or with suitable buffers, lysed, and the protein isolated by columnchromatography, for example, on DEAE-cellulose, phosphocellulose,polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis orimmunoprecipitation. Alternatively, the TSG-6 or functional derivativethereof may be isolated by the use of anti-TSG-6 antibodies. Suchantibodies may be obtained by well-known methods, some of which arementioned below.

[0165] Genetic constructs encoding TSG-6 functional derivatives thereofsuch as those described above, can be used in gene therapy. An abnormalTSG-6 molecule which results in enhanced susceptibility to disease, maybe replaced by infusion of cells of the desired lineage (such asfibroblasts, for example) transfected with DNA encoding normal ormodified TSG-6 protein, under conditions where the infused cells willpreferentially replace the endogenous cell population.

[0166] The present invention is also directed to a transgenic non-humaneukaryotic animal (preferably a rodent, such as a mouse) the germ cellsand somatic cells of which contain genomic DNA according to the presentinvention which encodes the TSG-6 protein or a functional derivativethereof. The TSG-6 DNA is introduced into the animal to be madetransgenic, or an ancestor of the animal, at an embryonic stage,preferably the one-cell, or fertilized oocyte, stage, and generally notlater than about the 8-cell stage. The term “transgene,” as used herein,means a gene which is incorporated into the genome of the animal and isexpressed in the animal, resulting in the presence of protein in thetransgenic animal.

[0167] There are several means by which such a gene can be introducedinto the genome of the animal embryo so as to be chromosomallyincorporated and expressed. One method is to transfect the embryo withthe gene as it occurs naturally, and select transgenic animals in whichthe gene has integrated into the chromosome at a locus which results inexpression. Other methods for ensuring expression involve modifying thegene or its control sequences prior to introduction into the embryo. Onesuch method is to transfect the embryo with a vector (see above)containing an already modified gene. Other methods are to use a gene thetranscription of which is under the control of a inducible orconstitutively acting promoter, whether synthetic or of eukaryotic orviral origin, or to use a gene activated by one or more base pairsubstitutions, deletions, or additions (see above).

[0168] Introduction of the desired gene sequence at the fertilizedoocyte stage ensures that the transgene is present in all of the germcells and somatic cells of the transgenic animal and has the potentialto be expressed in all such cells. The presence of the transgene in thegerm cells of the transgenic “founder” animal in turn means that all itsprogeny will carry the transgene in all of their germ cells and somaticcells. Introduction of the transgene at a later embryonic stage in afounder animal may result in limited presence of the transgene in somesomatic cell lineages of the founder; however, all the progeny of thisfounder animal that inherit the transgene conventionally, from thefounder's germ cells, will carry the transgene in all of their germcells and somatic cells.

[0169] Chimeric non-human mammals in which fewer than all of the somaticand germ cells contain the TSG-6 DNA of the present invention, such asanimals produced when fewer than all of the cells of the morula aretransfected in the process of producing the transgenic mammal, are alsointended to be within the scope of the present invention.

[0170] The techniques described in Leder, U.S. Pat. No. 4,736,866(hereby incorporated by reference) for producing transgenic non-humanmammals may be used for the production of the transgenic non-humanmammal of the present invention. The various techniques described inPalmiter, R. et al, Ann. Rev. Genet. 20:465-99 (1986), the entirecontents of which are hereby incorporated by reference, may also beused.

[0171] The animals carrying the TSG-6 gene can be used to test compoundsor other treatment modalities which may prevent, suppress or curechronic inflammatory conditions mediated by TNF action on connectivetissue cells. These tests can be extremely sensitive because of theability to adjust the dose of an agent under test given to thetransgenic animals of this invention. Such diseases include, but are notlimited to rheumatoid arthritis, granulomatous diseases, and the like.Transgenic animals according to the present invention can also be usedas a source of cells for cell culture.

[0172] This invention is also directed to an antibody specific for anepitope of TSG-6 protein. In additional embodiments, the antibodies ofthe present invention are used in methods to detect the presence of, ormeasure the quantity or concentration of, TSG-6 protein in a cell, or ina cell or tissue extract, or a biological fluid. The antibodies may alsobe used in methods for measuring induction of expression of TSG-6 in acell or in methods for identifying a compound capable of inducing theexpression of TSG-6 in a cell. The antibodies may also be used todisrupt the action of TSG-6, thereby preventing or treating diseasesassociated with overproduction, or inappropriate production or action ofTSG-6, such as inflammatory disorders including rheumatoid arthritis,infections and sepsis, as well as conditions associated withTNF-stimulated leukocyte adhesion.

[0173] The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, and anti-idiotypic(anti-Id) antibodies.

[0174] An antibody is said to be “capable of binding” a molecule if itis capable of specifically reacting with the molecule to thereby bindthe molecule to the antibody. The term “epitope” is meant to refer tothat portion of any molecule capable of being bound by an antibody whichcan also be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

[0175] An “antigen” is a molecule or a portion of a molecule capable ofbeing bound by an antibody which is additionally capable of inducing ananimal to produce antibody capable of binding to an epitope of thatantigen. An antigen may have one, or more than one epitope. The specificreaction referred to above is meant to indicate that the antigen willreact, in a highly selective manner, with its corresponding antibody andnot with the multitude of other antibodies which may be evoked by otherantigens.

[0176] In order to predict antigenic epitopes present in the protein,the amino acid sequence is inspected visually or analyzed by computer,for example, using the program of PEPTIDESTRUCTURE (Jameson et al,CABIOS 4: 181-186 (1988)). This program allows determination ofhydropathicity values which are then used to determine which peptidesequences within the overall protein sequence are likely to be mostimmunogenic based on their potential secondary structure. Such peptidesmay be synthesized chemically, or alternatively, and preferably, byrecombinant DNA methods.

[0177] Such computer analysis of the sequence of the TSG-6 protein ledto the selection of three sequences each of 15 amino acids fromdifferent parts of the molecule. These synthetic peptides weresynthesized by the NYU Cancer Center Peptide Synthesis Laboratory. Onesequence was selected on the basis of its high degree of homology withproteoglycan core/cartilage link protein/CD44, whereas the other twopeptides were from other portions of the TSG-6 protein and showed nosignificant homology to other known proteins. A cysteine residue wasadded to the N- or C-terminus of each of the synthetic peptides tofacilitate coupling to keyhole limpet hemocyanin (KLH), to be used ascarrier protein. The 15-mers were coupled to KLH with the aid of theheterobifunctional reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS) as described (Hartlow, E. et al, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988))and employed for the immunization of rabbits, using 2 rabbits per eachsynthetic peptide-KLH conjugate.

[0178] One of the pitfalls of generating antibodies to syntheticpeptides is the possibility that an antibody so raised may fail to reactwith the native protein. For this reason, alternative approaches may beused. The TSG-6 protein may be prepared as a bacterially expressedfusion protein by using an appropriate expression plasmid (see Examples,below). The purified fusion protein is employed for the immunization ofrabbits. Alternatively, such a fusion protein, or a synthetic peptidemay be used to immunize a rodent for generation of a monoclonalantibody.

[0179] Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen.

[0180] Monoclonal antibodies are a substantially homogeneous populationof antibodies to specific antigens. MAbs may be obtained by methodsknown to those skilled in the art. See, for example Kohler and Milstein,Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110. Such antibodiesmay be of any immunoglobulin class including IgG, IgM, IgE, IgA, and anysubclass thereof. The hybridoma producing the mAbs of this invention maybe cultivated in vitro or in vivo. Production of high titers of mAbs invivo production makes this the presently preferred method of production.Briefly, cells from the individual hybridomas are injectedintraperitoneally into pristane-primed Balb/c mice to produce ascitesfluid containing high concentrations of the desired mAbs. MAbs ofisotype IgM or IgG may be purified from such ascites fluids, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

[0181] Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies and methods for their production are knownin the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277(1984); Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984);Boulianne et al, Nature 312:643-646 (1984); Neuberger et al, Nature314:268-270 (1985); Taniguchi et al, European Patent Application 171496(published Feb. 19, 1985); Kudo et al, European Patent Application184187 (published Jun. 11, 1986); Robinson et al, International PatentPublication #PCT/US86/02269 (published May 7, 1987); Sun et al, Proc.Natl. Acad. Sci. USA 84:214-218 (1987); Better et al, Science240:1041-1043 (1988)). These references are hereby incorporated byreference.

[0182] An anti-idiotypic (anti-Id) antibody is an antibody whichrecognizes unique determinants generally associated with theantigen-binding site of an antibody. An Id antibody can be prepared byimmunizing an animal of the same species and genetic type (e.g. mousestrain) as the source of the mAb with the mAb to which an anti-Id isbeing prepared. The immunized animal will recognize and respond to theidiotypic determinants of the immunizing antibody by producing anantibody to these idiotypic determinants (the anti-Id antibody).

[0183] The anti-Id antibody may also be used as an “immunogen” to inducean immune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may bear structural similarityto the original mAb which induced the anti-Id. Thus, by using antibodiesto the idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

[0184] Accordingly, mAbs generated against the TSG-6 protein of thepresent invention may be used to induce anti-Id antibodies in suitableanimals, such as Balb/c mice. Spleen cells from such immunized mice areused to produce anti-Id hybridomas secreting anti-Id mAbs. Further, theanti-Id mAbs can be coupled to a carrier such as keyhole limpethemocyanin (KLH) and used to immunize additional Balb/c mice. Sera fromthese mice will contain anti-anti-Id antibodies that have the bindingproperties of the original mAb specific for an TSG-6 protein epitope.

[0185] The term “antibody” is also meant to include both intactmolecules as well as fragments thereof, such as, for example, Fab andF(ab′)₂, which are capable of binding antigen. Fab and F(ab′)₂ fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding than anintact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)).

[0186] It will be appreciated that Fab and F(ab′)₂ and other fragmentsof the antibodies useful in the present invention may be used for thedetection and quantitation of TSG-6 protein according to the methodsdisclosed herein for intact antibody molecules. Such fragments aretypically produced by proteolytic cleavage, using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments).

[0187] The antibodies, or fragments of antibodies, of the presentinvention may be used to quantitatively or qualitatively detect thepresence of cells which express the TSG-6 protein on their surface orintracellularly. This can be accomplished by immunofluorescencetechniques employing a fluorescently labeled antibody (see below)coupled with light microscopic, flow cytometric, or fluorimetricdetection.

[0188] The antibodies of the present invention may be employedhistologically, as in immunofluorescence or immunoelectron microscopy,for in situ detection of TSG-6 protein. In situ detection may beaccomplished by removing a histological (cell or tissue) specimen from asubject and providing the a labeled antibody of the present invention tosuch a specimen. The antibody (or fragment) is preferably provided byapplying or by overlaying on the biological sample. Through the use ofsuch a procedure, it is possible to determine not only the presence ofthe TSG-6 protein but also its distribution on the examined tissue.Using the present invention, those of ordinary skill will readilyperceive that any of a wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

[0189] Additionally, the antibody of the present invention can be usedto detect the presence of soluble TSG-6 molecules in a biologicalsample. Used in this manner, the antibody can serve as a means tomonitor the presence and quantity of TSG-6 proteins in a subject havinga condition associated with TNF induction of TSG-6, such as aninflammatory condition, an infection or sepsis, and the like.

[0190] Such immunoassays for TSG-6 protein typically comprise incubatinga biological sample, such as a biological fluid, a tissue extract,freshly harvested cells such as lymphocytes or leucocytes, or cellswhich have been incubated in tissue culture, in the presence of adetectably labeled antibody capable of identifying TSG-6 protein, anddetecting the antibody by any of a number of techniques well-known inthe art.

[0191] The biological sample may be treated with a solid phase supportor carrier (which terms are used interchangeably herein) such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled TSG-6-specific antibody. The solid phase support may then bewashed with the buffer a second time to remove unbound antibody. Theamount of bound label on said solid support may then be detected byconventional means.

[0192] By “solid phase support or carrier” is intended any supportcapable of binding antigen or antibodies. Well-known supports, orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Those skilled inthe art will know many other suitable carriers for binding antibody orantigen, or will be able to ascertain the same by use of routineexperimentation.

[0193] The binding activity of a given lot of anti-TSG-6 antibody may bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

[0194] Other such steps as washing, stirring, shaking, filtering and thelike may be added to the assays as is customary or necessary for theparticular situation.

[0195] One of the ways in which the TSG-6-specific antibody can bedetectably labeled is by linking the same to an enzyme and use in anenzyme immunoassay (EIA). This enzyme, in turn, when later exposed to anappropriate substrate, will react with the substrate in such a manner asto produce a chemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

[0196] Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect TSG-6 protein through theuse of a radioimmunoassay (RIA) (Chard, T., “An Introduction toRadioimmune Assay and Related Techniques” (In: Work, T. S., et al,Laboratory Techniques in Biochemistry in Molecular Biology, NorthHolland Publishing Company, New York (1978), incorporated by referenceherein). The radioactive isotope can be detected by such means as theuse of a gamma counter or a liquid scintillation counter or byautoradiography. It is also possible to label the antibody with afluorescent compound. When the fluorescently labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0197] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0198] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0199] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0200] The antibody molecules of the present invention may be adaptedfor utilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportand a quantity of detectably labeled soluble antibody is added to permitdetection and/or quantitation of the ternary complex formed betweensolid-phase antibody, antigen, and labeled antibody.

[0201] Typical, and preferred, immunometric assays include “forward”assays in which the antibody bound to the solid phase is first contactedwith the sample being tested to “extract” the antigen from the sample byformation of a binary solid phase antibody-antigen complex. After asuitable incubation period, the solid support is washed to remove theresidue of the fluid sample, including unreacted antigen, if any, andthen contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support through the unlabeled antibody,the solid support is washed a second time to remove the unreactedlabeled antibody.

[0202] In another type of “sandwich” assay, which may also be usefulwith the antigens of the present invention, the so-called “simultaneous”and “reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support and labeledantibody are both added to the sample being tested at the same time.After the incubation is completed, the solid support is washed to removethe residue of fluid sample and uncomplexed labeled antibody. Thepresence of labeled antibody associated with the solid support is thendetermined as it would be in a conventional “forward” sandwich assay.

[0203] In the “reverse” assay, stepwise addition first of a solution oflabeled antibody to the fluid sample followed by the addition ofunlabeled antibody bound to a solid support after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support is then determinedas in the “simultaneous” and “forward” assays.

[0204] According to the present invention, it is possible to diagnosecirculating antibodies in a subject which are specific for the TSG-6protein. This is accomplished by means of an immunoassay, as describedabove, using the protein of the invention or a functional derivativethereof.

[0205] In cancer patients, circulating endotoxin levels are high(Harris, R. I. et al, J. Clin. Path. 37:467-470 (1984)), particularly inpatients with tumor types known to be associated with an increasedincidence of cachexia (Humber-stone, D. A. et al, Cancer 62:1619-1624(1988)). The presence of high endotoxin levels is probably not a directresult of the tumor per se, but rather reflects the general debility ofthe patients. Increased translocation from the gut of endogenousbacteria and endotoxins in critical illness is dependent on the presenceof malnutrition and that impaired cell-mediated immunity may be anaggravating factor (Wilmore, D. W. et al, Surgery 104:917-923 (1988)).As cachectic cancer patients are malnourished and often exhibitsuppression of cell-mediated immunity, translocation of endogenousorganisms may account for higher levels of endotoxins. Cancer patients'peripheral blood mononuclear cells often show enhanced “spontaneous” TNFrelease in vitro (Aderka, D. et al, Lancet i:1190-1192 (1985)). TNFproduction in response to macrophage-activating agents is reduced inpatients with advanced metastatic disease but not in cancer patientswith only localized disease. These observations supported the notionthat TNF production is ongoing in cancer patients, either due tosustained stimulation of monocytes/macrophages by tumor cells or todirect TNF production by tumor cells. TNF was detected in the serum of50% of 226 cancer patients with active disease, compared to 3% ofhealthy sera and 18% of sera from disease-free cancer patients(Balkwill, F. et al, Lancet ii: 1229-1232 (1987)).

[0206] TNF levels are also elevated in a variety of bacterial and viralillnesses, including AIDS (Lahdevirta, J. et al, Amer. J. Med.85:289-291 (1988)) and meningococcal meningitis and septicemia (Waage,A. et al, (Lancet i:355-357 (1987)). In a rat burn/infection model,levels of hepatic TNF mRNA increased 100% in rats subjected toburn+infection compared to controls or rats subjected to burns but noinfection (Marano, M. A. et al, Arch. Surg. 123:1383-1388 (1988)). Theanimals subjected to burn and infection also showed a greater metabolicresponse (cachexia). Michie, H. R. et al, Br. J. Surg. 76:670-671(1989), reviewed evidence that TNF is the principal mediator associatedwith the changes of severe sepsis.

[0207] Therefore, the methods of the present invention which are capableof measuring the response of a subject to a cytokine such as TNF orIL-1, or to bacterial endotoxin are useful in predicting thesusceptibility of that individual to the debilitating effects of canceror infectious disease. Similarly, the compositions of the presentinvention are useful in the prevention or treatment of such diseases,due to their ability to disrupt events set into motion by the action ofTNF.

[0208] As used herein, the term “prevention” of a condition, such as aninflammatory response, infectious disease or a malignant tumor, in asubject involves administration of the TSG-6 peptide derivative, orantibody (see above) prior to the clinical onset of the disease.“Treatment” involves administration of the protective composition afterthe clinical onset of the disease. For example, successfuladministration of a TSG-6 peptide derivative or anti-TSG-6 antibodyaccording to the invention after development of an inflammatorycondition, a malignant tumor or an infection comprises “treatment” ofthe disease.

[0209] The TSG-6 protein, peptides or antibodies-of the presentinvention may be administered by any means that achieve their intendedpurpose, for example, to treat rheumatoid arthritis or otherinflammatory conditions, malignant tumors, infections, and the like.

[0210] For example, administration may be by various parenteral routessuch as subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal, intranasal, transdermal, or buccal routes.Alternatively, or concurrently, administration may be by the topicalroute or the oral route. Parenteral administration can be by bolusinjection or by gradual perfusion over time.

[0211] A typical regimen for preventing, suppressing, or treating acondition such as chronic inflammation, as in rheumatoid arthritis, or amalignant tumor, comprises administration of an effective amount of theTSG-6 functional derivative, or an antibody thereto, administered over aperiod of one or several days, up to and including between one week andabout six months.

[0212] It is understood that the dosage administered will be dependentupon the age, sex, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired. The ranges of effective doses provided below are notintended to be limiting and represent preferred dose ranges. However,the most preferred dosage will be tailored to the individual subject, asis understood and determinable by one of skill in the art.

[0213] The total dose required for each treatment may be administered bymultiple doses or in a single dose. The protein, functional derivativethereof or antibody may be administered alone or in conjunction withother therapeutics directed to the viral infection, or directed to othersymptoms of the viral disease.

[0214] Effective amounts of the TSG-6 protein, functional derivativethereof, or antibody thereto, are from about 0.01 μg to about 100 mg/kgbody weight, and preferably from about 10 μg to about 50 mg/kg bodyweight.

[0215] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions, which maycontain auxiliary agents or excipients which are known in the art.Pharmaceutical compositions such as tablets and capsules can also beprepared according to routine methods.

[0216] Pharmaceutical compositions comprising the proteins, peptides orantibodies of the invention include all compositions wherein theprotein, peptide or antibody is contained in an amount effective toachieve its intended purpose. In addition, the pharmaceuticalcompositions may contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.

[0217] Pharmaceutical compositions include suitable solutions foradministration by injection or orally, and contain from about 0.01 to 99percent, preferably from about 20 to 75 percent of active component(i.e., the TSG-6 protein or antibody) together with the excipient.Pharmaceutical compositions for oral administration include tablets andcapsules. Compositions which can be administered rectally includesuppositories.

[0218] Chondroitin-sulfate-rich proteoglycan is an essential componentof the matrix of cartilage since it enables the tissue to resistcompression during load bearing. Loss of proteoglycan, such as occurs inrheumatoid arthritis, osteoarthritis and other joint diseases, resultsin severe impairment of the function of cartilage. TNF and IL-1 areknown to cause degradation of this proteoglycan in cartilage(Saklatvala, J. et al, Biochem. J. 224:461 (1984); J. Exp. Med. 162:1208(1985); Saklatvala, J., Nature 322:547 (1986)), and to inhibit itsre-synthesis. In the extracellular matrix, proteoglycan corenoncovalently binds to a hyaluronic acid chain through two link proteinsthat bind to both the core protein of the proteoglycan and to thehyaluronic acid chain, thereby stabilizing the aggregates.

[0219] The N-terminal half of TSG-6 protein has significant homologywith cartilage proteoglycan core protein and link protein. Thus,according to the present invention, in connective tissue, TNF or IL-1induces the secretion of TSG-6, which interacts with link proteins orthe proteoglycan core protein through a shared homology domain. TSG-6may compete either with link protein or with proteoglycan core proteinfor binding to the hyaluronic acid chain, resulting in destabilizationof the structure of the proteoglycan aggregates, which causesproteoglycan release.

[0220] The C-terminal half of TSG-6 has a high degree of homology withthe interaction domain of the complement C1r-A chain. The complementcomponents C1r and C1s (which interacts with C1r through the interactiondomain) are serine proteases. Therefore, upon binding to a hyaluronicacid chain via its N-terminal portion, TSG-6 may attract proteasesthrough its C1r-like domain. This results in the proteases causinglimited proteolysis of the proteoglycan. See FIG. 17 for a schematicillustration of this interaction.

[0221] Thus, according to the present invention, antibodies specific tothe N-terminal portion of the TSG-6 protein, or functional derivativesof TSG-6, preferably peptides from the N-terminal portion havinghomology to the proteoglycan core protein or to the cartilage linkprotein, are useful in inhibiting TNF-induced, TSG-6-mediated, breakdownof proteoglycan in cartilage. In addition, antibodies specific to theC-terminal portion of the TSG-6 protein, or functional derivatives ofTSG-6, preferably peptides from the C-terminal potion having homology toC1r, are useful in inhibiting TNF-induced, TSG-6-mediated, breakdown ofproteoglycan in cartilage. Therefore, according to the presentinvention, diseases involving proteoglycan breakdown, such as, forexample, rheumatoid arthritis and other inflammatory connective tissuedisorders, may be treated using antibodies specific for epitopes ofTSG-6 or peptides corresponding to portions of TSG-6 as described above.

[0222] Cartilage cells (chondrocytes) may be used for production ofTSG-6, or for evaluating the efficacy of treatments using TSG-6-specificantibodies or TSG-6 functional derivatives such as peptides. Cartilagecells are one of the family of connective tissue cells, and these cellsare highly responsive to the pro-inflammatory cytokines, TNF and IL-1.TNF and IL-1 stimulate resorption of cartilage (Saklatvala, J., supra),a major feature of inflammatory diseases such as rheumatoid arthritis.Furthermore, IL-6 is secreted by chondrocytes upon treatment with TNF orIL-1 (Guerne, P.-A. et al, supra) as part of an inflammatory process,similar to the response to FS-4 fibroblasts to these cytokines.

[0223] Cells are isolated from human articular cartilage by sequentialenzymatic digestion using standard procedures (Pieter, A. et al,Arthritis Rheum. 25:1217 (1982); Malejczyk, J. et al, Clin. Exp.Immunol. 75:477 (1989)). An exemplary method for preparing cartilagechips (1 cm²) from the superficial layers of cartilage, avoidingcalcified layers, follows. The cartilage chips are first treated withhyaluronidase (0.5 mg/ml for 15 min at room temperature) to remove anyother cell types that might potentially adhere to the cartilage surface,followed by five washes with PBS. The chips are then minced into smallpieces of about 0.5 mm³ and digested with collagenase (2 mg/ml) andDNase (0.1 mg/ml) in the presence of 10% FCS for 18 h at 37° C. in agyratory shaker. The resulting chondrocytes are cultured in 175-cm²tissue culture flasks in DMEM and 10% FCS. After 24 h, cells aredetached by gentle pipetting, washed, and recultured for 4 hours in thepresence of TNF (20 ng/ml) at a density of 5×10⁷ cells/75 cm2 flask.Total RNA is isolated from the TNF treated cells by the guanidinethiocyanate/hot phenol method (Feramisco, J. R. et al, J. Biol. Chem.257:11024 (1982)). Northern blot analysis is performed to test forinduction of TSG-6 mRNA; alternatively, antibodies specific for TSG-6can be used to test extracts or supernatants of chondrocytes forproduction of TSG-6 protein, as described herein.

[0224] The preferred animal subject of the present invention is amammal. By the term “mammal” is meant an individual belonging to theclass Mammalia. The invention is particularly useful in the treatment ofhuman subjects, although it is intended for veterinary uses as well.

[0225] The present invention provides methods for evaluating thepresence and the level of normal or mutant TSG-6 protein or mRNA in asubject. For example, over-expression of TSG-6 in response tostimulation with TNF or IL-1, or an exogenous stimulus such as abacterial infection, may serve as an important predictor of theinflammatory or septic response. By providing a means to measure thequantity of TSG-6 mRNA in a hybridization assay or to measure TSG-6protein, as in an immunoassay, the present invention provides a meansfor detecting susceptibility in a subject to development of aninflammatory condition, such as rheumatoid arthritis, to infectious andseptic conditions, and the like.

[0226] Oligonucleotide probes encoding various portions of the TSG-6 DNAsequence are used to test cells from a subject for the presence TSG-6DNA or mRNA. A preferred probe would be one directed to the nucleic acidsequence encoding at least 12 and preferably at least 15 nucleotides ofthe TSG-6 sequence. Qualitative or quantitative assays can be performedusing such probes. For example, Northern analysis (see Examples below)is used to measure expression of an TSG-6 mRNA in a cell or tissuepreparation.

[0227] Such methods can be used even with very small amounts of DNAobtained from an individual, following use of selective amplificationtechniques. Recombinant DNA methodologies capable of amplifying purifiednucleic acid fragments have long been recognized. Typically, suchmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by Cohen et al (U.S. Pat. No. 4,237,224),Sambrook et al (supra), etc.

[0228] Recently, an in vitro enzymatic method has been described whichis capable of increasing the concentration of such desired nucleic acidmolecules. This method has been referred to as the “polymerase chainreaction” or “PCR” (Mullis, K. et al, Cold Spring Harbor Symp. Quant.Biol. 51:263-273 (1986); Erlich H. et al, EP 50,424; EP 84,796, EP258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al, U.S. Pat.No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al,U.S. Pat. No. 4,683,194).

[0229] The polymerase chain reaction provides a method for selectivelyincreasing the concentration of a particular nucleic acid sequence evenwhen that sequence has not been previously purified and is present onlyin a single copy in a particular sample. The method can be used toamplify either single- or double-stranded DNA. The essence of the methodinvolves the use of two oligonucleotide probes to serve as primers forthe template-dependent, polymerase mediated replication of a desirednucleic acid molecule.

[0230] The precise nature of the two oligonucleotide probes of the PCRmethod is critical to the success of the method. As is well known, amolecule of DNA or RNA possesses directionality, which is conferredthrough the 5′-3′ linkage of the phosphate groups of the molecule.Sequences of DNA or RNA are linked together through the formation of aphosphodiester bond between the terminal 5′ phosphate group of onesequence and the terminal 3′ hydroxyl group of a second sequence.Polymerase dependent amplification of a nucleic acid molecule proceedsby the addition of a 5′ nucleotide triphosphate to the 3′ hydroxyl endof a nucleic acid molecule. Thus, the action of a polymerase extends the3′ end of a nucleic acid molecule. These inherent properties areexploited in the selection of the oligonucleotide probes of the PCR. Theoligonucleotide sequences of the probes of the PCR method are selectedsuch that they contain sequences identical to, or complementary to,sequences which flank the particular nucleic acid sequence whoseamplification is desired.

[0231] More specifically, the oligonucleotide sequence of the “first”probe is selected such that it is capable of hybridizing to anoligonucleotide sequence located 3′ to the desired sequence, whereas theoligonucleotide sequence of the “second” probe is selected such that itcontains an oligonucleotide sequence identical to one present 5′ to thedesired region. Both probes possess 3′ hydroxy groups, and therefore canserve as primers for nucleic acid synthesis.

[0232] In the PCR, the reaction conditions are cycled between thoseconducive to hybridization and nucleic acid polymerization, and thosewhich result in the denaturation of duplex molecules. In the first stepof the reaction, the nucleic acids of the sample are transiently heated,and then cooled, in order to denature any double-stranded moleculeswhich may be present. The “first” and “second” probes are then added tothe sample at a concentration which greatly exceeds that of the desirednucleic acid molecule. When the sample is incubated under conditionsconducive to hybridization and polymerization, the “first” probe willhybridize to the nucleic acid molecule of the sample at a position 3′ tothe sequence to be amplified. If the nucleic acid molecule of the samplewas initially double-stranded, the “second” probe will hybridize to thecomplementary strand of the nucleic acid molecule at a position 3′ tothe sequence which is the complement of the sequence whose amplificationis desired. Upon addition of a polymerase, the 3′ ends of the “first”and (if the nucleic acid molecule was double-stranded) “second” probeswill be extended. The extension of the “first” probe will result in thesynthesis of an oligonucleotide having the exact sequence of the desirednucleic acid. Extension of the “second” probe will result in thesynthesis of an oligonucleotide having the exact sequence of thecomplement of the desired nucleic acid.

[0233] The PCR reaction is capable of exponential amplification ofspecific nucleic acid sequences because the extension product of the“first” probe, of necessity, contains a sequence which is complementaryto a sequence of the “second” probe, and thus can serve as a templatefor the production of an extension product of the “second” probe.Similarly, the extension product of the “second” probe, of necessity,contains a sequence which is complementary to a sequence of the “first”probe, and thus can serve as a template for the production of anextension product of the “first” probe. Thus, by permitting cycles ofpolymerization, and denaturation, a geometric increase in theconcentration of the desired nucleic acid molecule can be achieved.Reviews of the PCR are provided by Mullis, K. B. (Cold Spring HarborSymp. Quant. Biol. 51:263-273 (1986)); Saiki, R. K., et al(Bio/Technology 3:1008-1012 (1985)); and Mullis, K. B., et al (Meth.Enzymol. 155:335-350 (1987)).

[0234] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLE I Preparation of cDNA Library from TNF-Treated FS-4 Cells andIsolation of TNF-Inducible cDNA

[0235] Materials

[0236]E. coli-derived recombinant human TNF (specific activity, 3×10⁷U/mg) was supplied by M. Tsujimoto of the Suntory Institute forBiomedical Research, Osaka, Japan. E. coli-derived recombinant humanIL-1α (specific activity, 1×10⁹ U/mg) was received from Alvin Stern andPeter Lomedico, Hoffmann-LaRoche, Inc., Nutley, N.J. E. coli-derivedhuman gamma interferon (IFN-τ) (specific activity, 2.1×10⁷ U/mg) wasprovided by Biogen, Cambridge, Mass. E. coli-derived human IFN-β(Betaseron, specific activity, 2×10⁸ U/mg) was obtained from TritonBiosciences, Alameda, Calif. Epidermal growth factor (EGF),platelet-derived growth factor (PDGF) and transforming growth factor-β(TGF-β) were purchased from Collaborative Research, INc., Bedford, Mass.Poly(I)-poly(C) was from P-L 0 Biochemicals, Inc., Milwaukee, Wis.N⁶-2′-O-dibutyl adenosine cyclic 3′,5′-monophosphate, cycloheximide,forskolin, 12-O-tetradecanoylphorbol 13-acetate (TPA), the calciumionophore A23187, and isobutylmethylxanthine were purchased from SigmaChemical Co., St. Louis, Mo. The pHe7 plasmid, used as a source ofinternal reference cDNA (Kaczmarek, L. et al, J. Cell Biol. 104:183-187(1987), was supplied by P. B. Sehgal, Rockefeller University, New York,N.Y.

[0237] Cell Culture

[0238] The human diploid FS-4 foreskin fibroblast cell line (Vilcek, J.et al, Proc. Natl. Acad. Sci. USA 70:3909-3913 (1973)) was used atpassage level 15 in all experiments. FS-4 cells were grown in Eagleminimal essential medium (E-MEM) supplemented with 6 mM HEPES, 3 mMTricine, 50 μg/ml gentamicin, and 5% heat inactivated (56° C., 30 min)fetal bovine serum (FBS; GIBCO Laboratories, Grand Island, N.Y.). Forexperiments, 4×10⁶ cells were seeded in 175 cm² Falcon flasks, incubatedat 37° C., and allowed to grow to confluence over 6 days. The confluentmonolayers were washed once with phosphate buffered saline andreplenished with E-MEM containing 0.25% FBS. The cultures were incubatedin this medium for another 72 h at 37° C. to let the cells becomequiescent and then treated with the appropriate agents, as specifiedherein.

Preparation of cDNA and Construction of cDNA Library

[0239] Total cytoplasmic RNA was isolated from quiescent FS-4 cellstreated for 3 h with TNF (10 ng/ml) as described previously (Lin, J.-X.et al, J. Biol. Chem. 262:11908-11911 (1987)). Poly(A)⁺ RNA was selectedby one cycle of binding to oligo(dT)-cellulose (type 7; P-LBiochemicals). Double stranded cDNA was made from 10 μg of poly(A)⁺using the cDNA synthesis system of Bethesda Research Laboratories,Gaithersburg, Md. The double stranded cDNA was methylated with EcoRImethylase and made blunt-ended with T4 DNA polymerase. EcoRI linkerswere ligated onto the cDNA, which was then restricted with EcoRI. Theresulting cDNA greater than 600 base pairs in size was fractionated andseparated from the linker fragments by Sepharose CL4B columnchromatography and ligated into the EcoRI site of lambda gt10. Thelibrary was packed in vitro with Gigapack packaging extract(Stratagene).

[0240] Differential Screening of the cDNA Library The lambda gt10 cDNAlibrary was plated on E. coli LE392 at a density of 1000 PFU/dish (150mm diameter). Nitrocellulose filters were used to prepare duplicateplaque lifts of each plats. Prehybridization and hybridization offilters with ³²P-labeled single-stranded cDNA probe were performed asdescribed (Leonard, D. G. B. et al, Molec. Cell. Biol. 7:3156-3167(1987)). Probes were synthesized by using the Bethesda ResearchLaboratories cDNA synthesis system with 10 μg poly(A)⁺ RNA. The firststrand synthesis reaction was adjusted to contain 0.5 mM each of DATP,dGTP, and dTTP, 0.1 mM dCTP, 100 μg/ml dactinomycin, and 200 μCi of[α-³²P] dCTP (3000 Ci/mmol; ICN Pharmaceuticals, Inc., Irvine, Calif.).After synthesis of the cDNA, the RNA was removed by incubation in 0.2MNaOH at 70° C. for 20 min. The reaction was neutralized with HCl and thecDNA was ethanol precipitated in the presence of 2M ammonium acetate.The pellet was suspended in 200 μl of TE (10 mM Tris HCl, pH 8.0, 1 mMEDTA) and added to the hybridization solution and filters. One of twoprobes was used to hybridize to each of the two replica filters; one wasmade from untreated FS-4 cells, and the other was made from FS-4 cellstreated for 3 h with TNF (10 ng/ml). After hybridization, the filterswere washed in 2×SSC (1×SSC is 0.15M NaCl plus 0.015M sodiumcitrate)−0.1% sodium dodecyl sulfate (SDS) at 65° C. for 1 h with one ortwo changes. Filters ere exposed to Kodak XAR-5 film for 1-2 days withan intensifying screen at −70° C. Plaques that showed differentintensities of the hybridization signal with the two probes wereselected. these clones were subjected to one further round ofdifferential screening, and the plaques were purified.

[0241] Subcloning of the cDNA, Inserts, and Cross Hybridization Studies

[0242]E. coli LE392 cells in soft 0.7% agarose were poured into 150 mmplates. The lambda clones were then spotted on the plates in a gridarray. Four nitrocellulose filters were lifter from each plate,processed, and stored until use. To prepare cDNA inserts fromplaque-purified recombinant clones, 10 ml of liquid lysate was clarifiedand digested with 2 μg of DNase I per ml to remove contaminatedchromosomal DNA. Then 2 ml of 2.5% SDS-0.5M Tris HCl (pH9.5)−0.25M EDTAwas added, and plates were incubated at 65° C. for 15 min to lyse thephages. The solution was then cooled to room temperature before 2.0 mlof 10M ammonium acetate was added. The sample was chilled on ice for 20min and centrifuged at 15,000×g at 4° C. for 10 min to obtain the DNApellet. The pellet was suspended in 100 μl TE buffer containing 2 μgRNase A (Boehringer) per ml, and cut with the restriction enzyme EcoRI.The cDNA insert was isolated and subcloned into the EcoRI site of anM13mp19 vector. The cDNA inserts to be used as probes forcross-hybridization and Northern (RNA) blot experiments were preparedfrom the recombinant M13 clones by restriction with EcoRI to minimizebackground. The probes were prepared earlier. The hybridizationconditions ere essentially as described above for the differentialscreening experiments.

[0243] Northern Blot Analysis

[0244] Cytoplasmic RNA was fractionated on a 1% agarose gel containingformaldehyde and blotted onto Zeta-probe blotting membranes (Bio-RadLaboratories, Richmond, Calif.). Cytoplasmic RNA was loaded at 10μg/lane. Prehybridization and hybridization of Northern blots wereperformed as described (Lin, J.-X. et al, supra). Filters were probedwith 32P-labeled cDNA insert from recombinant M13 clones and/or with32P-labeled internal reference pHe7 insert. Northern blots werequantified with a laser densitometer.

[0245] Sequence Analysis

[0246] Single stranded DNA templates from recombinant M13 clones wereprepared, and several hundred nucleotides from each end of the cDNA weredetermined by the dideoxynucleotide-chain termination method (Sanger, F.et al, Proc. Natl. Acad. Sci. USA 74:5463-5468 (1977)). The partialnucleotide sequences were compared with sequences entered in GenBank(release 60.0).

[0247] Results

[0248] FS-4 cells were grown to confluence, then switched to medium with0.25% fetal bovine serum (FBS) and incubated for 72 h at 37° C. Thecells were then exposed to recombinant human TNF (10 ng/ml). CytoplasmicRNA was isolated (Lin, J.-X. et al, J. Biol. Chem. 262, 11908, 1987)after a 3-h incubation with TNF. A 3 h incubation with TNF was chosenfor the following reason. It is known that TNF induces an increase inthe level of some mRNAs within 20-30 min in quiescent FS-4 cells, butsome of these “early response” mRNAs are elevated only transiently, for30-120 min (e.g., c-fos and c-myc mRNA; see Lin, J.-X. et al, supra).Although such immediate early response gene products may be importantfor turning on other genes, the fact that they are induced onlytransiently suggested that they are not the actual effector moleculesresponsible for the phenotypic changes induced by TNF. Therefore, asearch was initiated for cDNAs corresponding to messages that are morestably elevated after TNF treatment.

[0249] Poly(A)+RNA was isolated from the cytoplasmic RNA by anddouble-stranded cDNA was synthesized. The resulting cDNA library fromTNF-treated FS-4 cells, consisting of 2×10⁶ recombinant clones, wasscreened for TNF-inducible gene sequences by differential hybridizationwith [³²P] cDNA probes prepared from poly(A)+RNA from control and fromTNF-treated FS-4 cells. Plaques which gave a strong signal when probedwith cDNA from TNF-treated cells, but not when probed with control cellcDNA, were picked as presumptive TNF-inducible genes.

[0250] Approximately 3×10⁴ plaques were screened, and 47 were scored asclearly inducible after two rounds of screening. They were designatedTSG 1-47 (TSG=“TNF-stimulated gene sequence”). To determine the numberof different mRNAs represented among the TSG clones selected bydifferential screening, the inserts were tested for sequence homology bycross-hybridization. A total of 44 cloned cDNAs have been examined bycross-hybridization to each other. These experiments revealed a total ofeight distinct, non-crossreacting cDNAs. As summarized in Table 2,below, some of the cDNAs were represented among the 44 clones with ahigh frequency (TSG-8 and TSG-14) while others were much less abundant(TSG-21, TSG-27 and TSG-37). The size of the corresponding mRNAs rangedfrom 0.8 to 4.5 kb. TABLE 2 Abundance of Individual TSG cDNAs Among 44TNF-Specific cDNA Clones Approximate Size of cDNA Abundance^(a)Corresponding mRNA (kb)  TSG-1  6 1.6  TSG-6  6 1.5  TSC-8 11 1.1 TSG-12 3 4.5 TSG-14 13 2.3 TSG-21  1 2.4 TSG-27  2 2.4 TSG-37  2 0.8

EXAMPLE II Kinetics of Induction of TNF-Induced mRNAs

[0251] To ascertain that the eight distinct TSG cDNAs isolated indeedcorrespond to mRNAs whose levels are up-regulated in FS-4 cells by TNFtreatment, quiescent FS-4 cultures were treated with TNF (20 ng/ml) fordifferent intervals ranging from 0.5 to 16 h, cytoplasmic RNA wasisolated (Lin, J.-X. et al, supra) and mRNA levels corresponding to eachof the eight cDNAs were quantitated by Northern blot analysis anddensitometric scanning of the autoradiograms (FIGS. 1 and 2). Theincrease in mRNA levels ranged from about 3-fold (TSG-21) to over100-fold (TSG-6 and TSG-8).

[0252] Three different patterns of mRNA stimulation were noted. Thefirst pattern was characterized by an increase to peak levels by 2-4 h,followed by a gradual decrease in mRNA levels (TSG-1 and TSG-6). Thesecond pattern showed a rapid increase of mRNA levels to a maximum by1.5 to 4 h, followed by a plateau until at least 16 h (TSG-8, TSG-12,TSG6-14 and TSG-37). The third pattern was characterized by a possibleinitial decrease, followed by a slow gradual increase in mRNA levelsthroughout the 16-h observation period (TSG-21 and TSG-27).

EXAMPLE III Partial Sequencing of TSG cDNAs

[0253] To determine whether the isolated cDNAs were homologous topreviously identified genes, all eight cDNAs were partially sequenced(300-400 bp) and the sequences determined were checked against sequencesavailable in GenBank. Sequences of five cDNAs (TSG-1, TSG-8, TSG-21,TSG-27 and TSG-37) were found to be identical to earlier identifiedgenes. Of these, TSG-1 corresponded to the gene forβ-thromboglobulin-like protein 3-10C (Schmid, J. et al, J. Immunol.139:250 (1987)), also known as IL-8. TSG-8 was identical to the recentlycloned gene for “monocyte chemotactic and activating factor” (MCAF)(Matsushima, K. et al, Cytokine 1:2 (1989)). TSG-21 and TSG-27 werefound to be identical to the collagenase and stromelysin genes,respectively, and TSG-37 was found to encode metallothionein II. Theother three partial cDNA sequences showed no significant homologies withknown genes, indicating that they represented hitherto unidentified genesequences.

[0254] Induction of IL-8 (=TSG-1) by TNF and by IL-1 was recentlyobserved by others (Matsushima, K. et al, supra; J. Exp. Med. 167:1883(1988)). IL-8, a neutrophil chemotactic factor, is structurally relatedto several members of a family of inflammatory cytokines that includeplatelet factor-4 (PF-4), the IFN-τ-inducible protein IP-10, thePDGF-inducible gene JE, proteins termed MIP-1 and MIP-2, and GRO(Matsushima, K. et al, supra; Larsen, C. G. et al, Science 243:1464(1989)). Most of these proteins appear to be chemotactic.

[0255] MCAF (=TSG-8) induction in human fibroblasts by TNF and IL-1 hasbeen recently described (Matsushima, K. et al, supra). Interestingly,MCAF shows significant amino acid sequence similarity (21%) with IL-8,and they both have four cysteines at similar positions.

[0256] Collagenase (=TSG-21) was also reported earlier to beTNF-inducible in synovial cells and fibroblasts (Dayer et al, supra). Itis very likely that the ability of TNF to induce collagenase is relatedto TNF's role in tissue remodeling during inflammation. While theinduction of stromelysin by TNF has not been reported, stromelysin mRNAwas recently shown to be inducible by IL-1 (Quinones, S. et al, J. Biol.Chem. 264:8339 (1989)). Like collagenase, stromelysin is acollagen-degrading metalloproteinase, and both can also degradefibronectin, laminin and cartilage proteoglycans. Both collagenase andstromelysin are thought to be important in the increased extracellularmatrix degradation occurring in rheumatoid arthritis.

[0257] Finally, metallothionein II (MT-II) (=TSG-37) has been shown tobe inducible by various stresses, including heavy metal challenge,injection of lipopolysaccharide as well as by cytokines includinginterferons and IL-1 (Karin, M., Cell 41:9-10 (1985)). In addition toits ability to bind heavy metal ions, MT-II may also act as a scavengerof free radicals released by activated macrophages and neutrophilsduring an inflammatory response. MT-II induction would thus serve aprotective role in the prevention of tissue injury (Thornalley, P. J.,Biochim. Biophys. Acta 827:36-44 (1985).

[0258] It is significant that all five TSG cDNAs identified bysequencing correspond to genes coding for proteins important in theinflammatory process. These results support the utility of the presentapproach of cloning TNF-inducible cDNAs from human fibroblasts in theidentification of novel genes with important functions in immuneresponses and inflammation.

EXAMPLE IV Patterns of TSG mRNA Induction by Different Cytokines andOther Agents

[0259] Table 3 provides a summary of a large number of experiments inwhich levels of mRNAs corresponding to the 8 distinct TSG cDNAs weredetermined in FS-4 cells exposed to various agents by Northern blotanalysis. Three of the mRNAs (corresponding to TSG-8, TSG-12 and TSG-14)were inducible by the protein synthesis inhibitor, cycloheximide (CHX).In some cases the addition of CHX either did not block (TSG-12 andTSG-14) or increased (TSG-1, TSG-6 and TSG-8) mRNA inducibility by TNF,suggesting that the increase in the mRNA levels is the result of adirect action of TNF, not requiring a protein intermediate. In contrast,induction of the remaining 3 mRNAs by TNF was inhibited or reduced inthe presence of CHX. Five of the mRNAs were inducible by IFN-β, but onlytwo of these responded to IFN-τ. Simultaneous treatment with IFN-βreduced inducibility of TSG-1 (=IL-8) mRNA by TNF, but increased theinducibility of TSG-6 and TSG-37 mRNAs. All mRNAs were inducible by IL-1and by the double-stranded RNA poly(I).poly(C) or the phorbol ester12-O-tetradecanoyl phorbol 13-acetate (TPA), but the efficiency ofinduction by these agents varied. Epidermal growth factor (EGF) wasmoderately efficient in increasing TSG-21 (=collagenase) and TSG-27(=stromelysin) mRNA levels, and weakly stimulated several other mRNAs.PDGF and transforming growth factor-β (TGF-β) were only weaklyeffective, as were dibutyryl cyclic AMP (dBcAMP) and thephosphodiesterase inhibitor, isobutyl methyl xanthine (IBMX).

[0260] As summarized in Table 3, it is apparent that none of the mRNAsresponded exclusively to TNF. However, at least two mRNAs (TSG-1/IL-8and TSG-6) were particularly strongly induced by TNF compared to anyother stimulus. It is interesting that TSG-1/IL-8 and TSG-6 mRNAs havesimilar patterns of inducibility (except for an apparent difference inthe actions of IFN-β and IFN-τ on induction by TNF). It is also apparentthat the pattern of inducibility of TSG-8 closely resembled that ofTSG-12; TSG-21 (collagenase) and TSG-27 (stromelysin) mRNA also hadsimilar patterns of inducibility. TSG-37 mRNA (metallothionein II) wasstrongly inducible by both TNF and IFN-β. TABLE 3 Effect of VariousTreatments on TSG mRNA levels in FS-4 Cells Relative Increase in mRNALevel TSG-1 TSG-8 TSG-21 TSG-27 TSG-37 Treatment^(a) (IL-6) TSG-6 (MCAF)TSG-12 TSG-14 (Collagenase) (Stromelysin) (MT-II) TNF +++ +++ +++ ++++++++ ++ ++ +++ CHX 0 0 + +++ + 0 0 0 TNF + CHX ++++ ++++ ++++ ++++ ++++0 0 + IFN-β 0 0 ++ + 0 ++ ++ +++ IFN-γ 0 0 ++ + 0 0 0 0 TNF + IFN-β +++++ +++ ++++ ++++ ++ ++ ++++ TNF + IFN-γ + +++ ++++ ++++ ++++ ++ ++ +++IL-1 + + ++ ++++ ++++ ++ ++ ++ EGF 0 0 + + + ++ ++ + PDGF 0 0 + + + 0 00 TGF-β 0 0 0/+ 0 0 0 0 0 Poly(I) .poly(C) + + +++ ++ ++ ++ ++ + TPA + +++ ++ + ++++ ++++ + A23187 + + ++ ++ 0 ND ND 0 Forskolin 0 0/+ 0 0 0 0 00 dBcAMP 0 0 + + 0 ND ND 0/+ IBMX 0 0/+ + + 0 0 0 0 # a mixture of TNFand CHX were toxic, TSG-21 and -27 mRNA levels in the groups treatedwith TNF and CHX were determined 4 h after the onset of treatment.)Total cellular RNA was isolated and fractionated on a 1% agarose gelcontaining formaldehyde; the RNA was then subjected to Northern blotanalysis as described in the Materials and Methods. Relative increasesin each of the TSG mRNA levels were quantitated by denaltometricscanning of the # autoradiograms: 0 indicates no increase in the mRNAlevel compared to the control mRNA level (no treatment); +, ++, +++,++++ indicate relative increase in the mRNA level after treatment ascompared with untreated control. For each of the individual TSG mRNAspecies, the highest denalty band(s) was (were) assigned ++++, and therelative denalties of other mRNAs bands in the same experiment were thenscored accordingly. ND, not determined.

EXAMPLE V Complete DNA Sequence of TSG-6 and Homology of the Protein toCD44/Hermes and the Cartilage Link Protein Family

[0261] Of the three TSG cDNAs having novel partial sequences, TSG-6 wasselected for further sequencing. Northern blot analysis showed that theTSG-6 cDNA hybridized to a single TNF-inducible mRNA band with anapparent size of 1.5 kb. Among the six lambda clones whichcross-hybridized with TSG-6 cDNA, the lambda5-TSG-6 clone had thelongest insert, of about 1.4 kb, and was therefore used for sequenceanalysis. The approximately 1.4 kb lambda5-TSG-6 insert was subclonedinto the EcoRI site of M13mp18 bacteriophage in both orientations. Toinsure fidelity of sequence determination, directional deletion cloneswere generated by the ExoIII/S1 method (Henikoff, S. supra) in bothdirections within the M13 clones. The deletion clones were then used todetermine the nucleotide sequence by the dideoxynucleotide chaintermination method (Sanger, F. et al, supra).

[0262] The TSG-6 cDNA was found to comprise 1414 bases, apparentlyconsisting of a 69-base 5′ untranslated region, a continuous openreading frame of 831 bases and a 3′ untranslated region (FIGS. 3A-3C).Within the 3′ untranslated region, there were multiple AT-rich regions.The corresponding mRNA sequence AUUUA is thought to confer instability(Shaw, G. et al, supra), resulting in rapid message degradation, and mayexplain the decline in the TSG-6 mRNA level seen after 4 h of continuoustreatment with TNF (see FIG. 1). A consensus polyadenylation signal(AATAAA) was also located at the 3′ end.

[0263] The largest open reading frame predicted a polypeptide of 277amino acids. No other open reading frame with a significant length wasfound. The putative initiation methionine codon is followed by 11consecutive hydrophobic amino acids followed by a charged region,suggesting a typical cleavable signal peptide (FIGS. 3A-3C and 4). Inaddition, the predicted TSG-6 protein sequence contains two potentialsites of N-linked glycosylation, and one potential chondroitin sulfatelinkage site (FIGS. 3A-3C and 4).

[0264] Comparison of the deduced amino acid sequence from TSG-6 cDNAwith the protein sequences available in databases revealed interestingsequence homologies. FIGS. 5A-5B show that the N-terminal motif of theTSG-6 gene product (between residues 37 and 127) has a high degree ofhomology with rat cartilage link protein (35.8% identity), ratproteoglycan core protein (38.9% identity) and the recently publishedsequence (Stamenkovic, I. et al, supra; Goldstein, L. A. et al, supra)of the human lymphocyte homing receptor CD44/gp90 Hermes (32.6%identity). In addition, the C-terminal portion of the TSG-6 gene productshows approximately 30% sequence homology with the α-fragment of thecomplement component C1r A chain.

[0265] The homology between a portion of the predicted sequence of theprotein encoded by TSG-6 cDNA and the CD44/Hermes family is ofparticular interest. The CD44/Hermes membrane proteins have beenimplicated in the lymph node “homing” of lymphocytes and their bindingto a variety of other tissues (Stoolman, L. M., supra). The fact thatCD44/Hermes is expressed in many hematopoietic, mesenchymal andepithelial cell lines suggests that this protein functions as amultipurpose adhesion receptor. The striking homology betweenCD44/Hermes and two repeated domains of cartilage link protein as wellas a domain of the proteoglycan core protein has been noted recently(Stamenkovic, I. et al, supra; Goldstein, L. A. et al, supra). Incartilage link protein and proteoglycan core protein these homologousregions are thought to be involved in the binding of these proteins bothto hyaluronic acid and to other proteoglycans through protein-proteininteractions. The presence of this epitope in CD44/Hermes may be relatedto the importance of cellular matrix interactions in lymphocyte traffic.

[0266] The highest degree of homology of any TSG-6 region and a knownsequence (about 60%) is to the hyaluronic acid binding sites of linkprotein and proteoglycan core protein. Based on the predicted secondarystructure, this region appears to be part of an extended loop formed bydisulfide bonds that are highly conserved in all of these proteins(FIGS. 5A-5B). Furthermore, the presence of a potential chondroitinsulfate linkage site suggests that the TSG-6 protein itself may be aso-called “part-time” proteoglycan (Ruoslahti, E., supra), as is alsothe case with CD44/Hermes.

[0267] A secreted form of the TSG-6 protein would be expected to play arole in leukocyte traffic and/or chemotaxis. The TSG-6 protein would bepredicted to bind to structures on the surface of leukocytes and altertheir adhesion characteristics and/or other function. TSG-6 protein islikely to recognize the same ligand as CD44/Hermes, i.e. hyaluronic acidand, possibly, other structures; in this event, soluble TSG-6 ispredicted to interfere with lymphocyte adhesion mediated by theCD44/Hermes molecule. Soluble TSG-6 is also predicted to inhibit celladhesion to the extracellular matrix.

[0268] The TSG-6 protein may also be a cell surface, membrane-associatedprotein, wherein its expression on the surface of fibroblasts (or othercells) would play a role in leukocyte adhesion. Cell-surface expressionof TSG-6 protein would alter the adhesion properties of the cellsproducing it to extracellular matrix molecules. Thus, TSG-6 expressionmay be related to the well-documented ability of TNF to either stimulateor inhibit cell growth, and to alter cellular morphology. In thisregard, it is interesting to note that TGF-β, another growth-modulatorycytokine, stimulates the synthesis of various proteoglycans (Bassols, A.et al, supra), including decorin (a secreted proteoglycan, a portion ofwhich remains cell surface-associated); this action could be directlyrelated to changes in cell morphology and cell growth characteristics(Ruoslahti, E., supra; Bassols, A. et al, supra). Like decorin, theTSG-6 polypeptide has a molecular weight of approximately 30 kDa and asingle chondroitin sulfate linkage site. Several membrane proteoglycans(including the murine homologue of CD44/Hermes) are known to interactwith the cytoskeleton, providing another potential mechanism wherebyincreased expression of the TSG-6 protein could lead to changes in cellgrowth or morphology.

[0269] It is therefore apparent that TSG-6 (either secreted orcell-associated) could have profound effects on vital properties of cellincluding cell growth, cell motility and cell-to-cell interactions. Inview of the ability of TSG-6 to bind hyaluronic acid (see below) and thefact that hyaluronic acid is an important component of both cellsurfaces and extracellular matrices, soluble TSG-6 would be postulatedto inhibit cell adherence. Cell adherence, both to other cells and tothe extracellular matrix, is important in the ability of malignant tumorcells to metastasize to distant sites in the body. One usefulapplication of TSG-6, in particular TSG-6 prepared by recombinant DNAtechnology, is as a prophylactic or therapeutic agent capable ofsuppressing the metastasis of tumor cells.

[0270] The homology between the C-terminal half of the TSG-6 protein andcomplement component C1r (FIG. 5B), is to a domain of C1r thought to bethe Ca²⁺ binding region, responsible for interaction between componentsC1r and C1s. This suggests that the homologous region of TSG-6 may be aCa²⁺ binding region and, therefore, perhaps involved in protein-proteininteractions.

EXAMPLE VI Preparation of Bacterial Fusion Proteins

[0271] To express a bacterial fusion protein of TSG-6, we used EcoRIcDNA insert from clone lambda6. Clone lambda6 contains a cDNA insertthat lacks 402 bp at the 5′-end and 4 bp at the 3′-end of the TSG-6 cDNAsequence shown in FIGS. 3A-3B. An EcoRI-BamH1 (406 bp) restrictionfragment (that encodes the portion of TSG-6 open reading frame spanningfrom Ile115 to Asp248) was isolated from the EcoRI cDNA insert of clonelambda6 and was cloned into the same restriction sites in the polylinkerdownstream of, and in frame with, a portion (35-37 kDa) of the E. coliTrpE open reading frame in the pATH21 vector (Sprindler, K. R. et al, J.Virol. 49:132-141 (1984)), resulting in the TrpE/TSG-6 expressionplasmid, pATH-TSG-6 (FIG. 6A)

[0272] The same restriction fragment (EcoRI-BamH1) was also insertedinto pEX34A bacterial expression vector, resulting in the MS2/TSG-6expression plasmid, pEX-TSG-6 (FIG. 6B). pEX34A is a derivative of pEX29(Klinkert, M. et al, Infec. Immun. 49:329-335 (1985)) which permits theproduction of foreign proteins fused to the N-terminal part of the MS2polymerase and controlled by the temperature-inducible PL promoter ofbacteriophage lambda.

[0273] Expression plasmid pATH-TSG-6 was transferred into competent E.coli HB101 cells. Transformed cells in M9 medium containing 2% Casaminoacids, 20 μg/ml of L-tryptophan, and 150 μg/ml of ampicillin were grownto a density of A₆₀₀=0.5 (absorbance at 600 nm). To induce synthesis ofthe fusion protein, cells were pelleted and resuspended in prewarmedL-tryptophan-free medium. After an additional 1-hour incubation, 20μg/ml of 3-β-indoleacrylic acid was added and the incubation wascontinued for an additional 24 hours. FIG. 7 shows that protein of theexpected size (approx. 54 kDa) was in fact induced following addition of3-β-indoleacrylic acid.

[0274] For expression of another TSG-6 fusion protein (MS2/TSG-6),recombinant plasmid pEX-TSG-6 was transferred into competent E. coli K12*H*Trp (Remaut, E. et al, Gene 15:81-03 (1981)), which contain atemperature-sensitive repressor of the lambdaPL promoter. Cells weregrown under selective conditions at 28° C. to high density. To inducesynthesis of the MS2/TSG-6 fusion protein, cells were diluted with 4volumes of prewarmed culture medium (LB) without antibiotics and thenincubated for 3 hours at 42° C. under good aeration. FIG. 8 shows that aMS2/TSG-6 fusion protein of the expected size (about 32 kDa) wasspecifically induced by high temperature.

[0275] Purification of both fusion proteins was done essentially asdescribed by Strebel et al (J. Virol. 57:983-991 (1985)). Cells from a1L culture were pelleted and washed with TEN (10 mM Tris-HCL, pH 8.0, 1mM EDTA, 0.5M NaCl), lysed with lysozyme (5 mg/ml) and finally broken bysonication. Insoluble material was recovered by centrifugation (30 min,20,000×g) and extracted sequentially with 20 ml of 3M urea and 5 ml of7M urea each for 30 min at 37° C. The 7M urea extract containing thefusion protein was further purified by preparative SDS-PAGE. Afterelectrophoresis the fusion protein was excised from the gel,electroeluted and concentrated as needed. The purity of theelectroeluted fusion protein was checked on analytical gels. After thesecond round of electroelution, highly purified fusion protein wasobtained with no detectable E. coli protein bands on SDS-PAGE (lane 7,in FIG. 7).

EXAMPLE VII Stable Transfection of TSG-6 cDNA into GM637 Cell Lines

[0276] The biological functions of TSG-6 can be studied by expression ofTSG-6 cDNA in a cell line which does not respond to TNF by the inductionof TSG-6 mRNA. For this purpose we examined the inducibility of TSG-6mRNA by TNF in the SV40 transformed GM637 cells, which do not expressTSG-6 mRNA upon treatment with TNF (see FIGS. 10A-10B). As aconstitutive expressor, an expression plasmid, pSV-TSG-6 (FIG. 9A) wasconstructed by replacing the β-tubulin isotype mβ1 coding region withthe full-length TSG-6 cDNA in the plasmid pSVβ1 (Lewis. S. A. et al,Cell 49:539-548 (1987)). In order to exploit suitable restriction enzymesites for easier cloning, we used the M13mp18 vector carrying thefull-length TSG-6 cDNA at the EcoRI site in either the sense orantisense orientation with respect to the lac promoter (Plac). AHindIII-NcoI fragment containing the 5′ region of TSG-6 cDNA wasisolated from the antisense construct and a NcoI-KpnI fragmentcontaining the 3′ region of TSG-6 cDNA was isolated from the senseconstruct. Both fragments were ligated into the HindIII/KpnI cleavedplasmid pSVβ1.

[0277] An inducible expression plasmid pMAM-TSG-6 (FIG. 9B) was alsoconstructed by ligating XbaI-NcoI fragment from the antisense constructand NcoI-SalI fragment from the sense construct into the NheI/SalIcleaved plasmid, pMAMneo (Sardet, C. et al, Cell 56:271 (1989)).Expression vector pMAMneo contains the RSV-LTR enhancer linked to thedexamethasone-inducible MMTV LTR promoter, a construction which yieldscontrollable high level expression of cloned genes in the presence ofdexamethasone. It also contains the E. coli neo gene, driven by the SV40later promoter, for selection of transfectants by growth in mediumcontaining the antibiotic G418.

[0278] Both constructs were used to transfect GM-637 cell lines by usingCaPO₄-DNA precipitation (Graham, F. L., Virology 52:456 (1973)). In thecase of stable transfection with pSV-TSG-6, pRSVneo (Gorman, C. et al,Science 221:551 (1983)), which confers resistance to G418, wascotransfected. For stable transfection of CaPO₄-DNA precipitates, GM-637cells were maintained in MEM containing 10% fetal calf serum andgentamycin sulfate at 37° C. and 5% CO₂ atmosphere. Cells were split 1:5with twice a week. To transfect human GM-637 cells, plates were seeded 1day prior to transfection at 5×10⁵ cells per 60 mm plate in 5 ml ofmedium. The TSG-6 expression plasmid, pMAM-TSG-6, or a mixture ofpSV-TSG-6 plus pRSVneo, was mixed with 0.25 ml of CaCl₂. An equal volumeof 2×HBS (pH 7.05) (280 mM NaCl, 10 mM HCl, 1.5 mM Na₂HPO₄.2H₂O, 12 mMdextrose, 50 mM HEPES) was added and the solution was incubated for 20min at room temperature. The CaPO₄-DNA suspension was dripped ontoplates and mixed. After 5 hr of incubating the cells at 37° C. in a 5%CO₂ atmosphere, the transfected cells were given a glycerol shock bytreatment with 15% glycerol in 1×HBS for 30 sec. and further incubatedfor 20 hr. The resulting tolerant cell monolayers were replated in themedium containing G418 (800 μg/ml) to select cells expressing theneomycin resistance marker. Colonies were isolated from transfectionplates via the use of cloning rings, subcloned in 24 well plates, andexpanded to monolayer culture. Multiple independent transfectants wereselected and tested for the expression of TSG-6 cDNA by northern blotanalysis.

[0279] FIGS. 10A-10B show that several transfectants express TSG-6 mRNAin the absence of TNF. The major band appears to be the same size as theband corresponding to TSG-6 mRNA induced by TNF in FS-4 cells. The upperbands may be the result of incomplete processing of TSG-6 cDNA in thepolyadenylation signal. The expression of TSG-6 protein in thesetransfectants was confirmed by Western blot analysis with the aid ofpolyclonal antiserum generated against the TSG-6 bacterial fusionprotein (see below).

EXAMPLE VIII Expression of TSG-6 mRNA by TNF: Specificity for NormalConnective Tissue

[0280] The inducibility of TSG-6 mRNA by TNF was examined in variouscell lines by Northern blot analysis. The presence of detectable TSG-6mRNA was of interest in view of the homology of TSG-6 protein to thehoming receptor, CD44. Human umbilical vein endothelial cells (HUVEC),which are one cell type shown to be highly responsive to TNF and usefulin the analysis of proinflammatory action of TNF, were examined. Alsoexamined were GM-637 cells, a line of SV40 virus-transformed humandiploid fibroblasts. FIG. 11 shows that these cell types do not expressTSG-6 mRNA upon TNF stimulation. Other selected cell lines (U937, A673,Colo205, HT29 and SK-MEL-19) also did not produce TSG-6 mRNA after TNFtreatment. This unresponsiveness is not due to a lack of TNF receptorson the cell surface because these cell lines have been shown to beresponsive to the actions of TNF (Le, J. et al, 1987, supra). Thesefindings indicate the possibility that the expression of TSG-6 mRNA isrestricted to cells of normal connective tissue origin.

[0281] To test this notion, the inducibility of TSG-6 mRNA in othernormal fibroblasts (FS-48, FS-49 and WI-38) and in fibroblast cell linestransformed with either SV40 virus (WI-38 VA13, GM-637) or SV40 large Tantigen only (FS-4-SV1, FS-4-SV2 and FS-4-SV3) was examined. TSG-6 mRNAwas indeed induced in all normal fibroblasts but not in SV40virus-transformed fibroblasts (WI-38 VA13 or GM-637). Interestingly, TNFinduction of TSG-6 mRNA was significantly decreased in FS-4 cellstransfected with SV40 large T antigen (FIG. 12). This was not due to thegeneral decrease in TNF responsiveness of these transfectants; forexample, TSG-14 mRNA, encoded by another TNF-stimulated gene (see above)is more highly induced in large T antigen transfectants than in FS-4cells after TNF treatment. It is concluded that the degree of“oncogenic” transformation has a controlling effect on the inducibilityof TSG-6 mRNA.

EXAMPLE IX Preparation of Polyclonal Antiserum and Purification ofAnti-TSG-6 Antibodies by Immunoaffinity Chromatography

[0282] Rabbits were first immunized with about 200 μg of the TrpE/TSG-6fusion protein suspended in Freund's complete adjuvant and were boostedat intervals of 2-3 weeks with the same amount of protein in Freund'sincomplete adjuvant. All injections were performed subcutaneously,except for the final boost which was done intravenously. Rabbits werebled six days after immunizations. Sera were analyzed by immunoblottingaccording to Strebel et al (supra).

[0283] Antibodies raised against the TrpE/TSG-6 fusion protein shownonspecific binding to a broad range of proteins from supernatants orextracts of FS-4 cells and other human cells. To obtain antibodiesspecific for TSG-6 domains of the fusion protein, the antiserum wassubjected to purification on an immunoaffinity matrix to which theMS2/TSG-6 fusion protein was coupled.

[0284] The immunoaffinity chromatography matrix was prepared as follows.Five mg of purified MS2/TSG-6 fusion protein was dialyzed extensivelyagainst 0.5M NaCl. Three ml of EAH-Sepharose 4B (Pharmacia) was washedextensively with 0.5M NaCl and the purified MS2/TSG-6 fusion protein wasadded. The pH was adjusted to 4.5 and 40 mg1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (AldrichChemical Co.) dissolved in 1 ml distilled water was added dropwise whileconstantly stirring. Thereafter, the pH was readjusted to 4.5 and thecoupling reaction was allowed to proceed overnight under constantstirring. Acetic acid (200 μl) was added for another 4 hr to block theremaining amino groups on the matrix. Finally, the matrix material waswashed several times alternately with 0.1M acetate buffer, pH 4.0, 0.5MNaCl, and 0.1M sodium bicarbonate buffer, pH 8.3, 0.5M NaCl, andsuspended in Tris-buffered saline for storage.

[0285] For immunoaffinity chromatography, 0.5 ml of MS2/TSG-6 Sepharosewas equilibrated with Tris-buffered saline (20 mM Tris, 0.5M NaCl, pH7.5) containing 0.05% Tween-20 (TTBS). One ml of antiserum raisedagainst the TrpE/TSG-6 fusion protein was mixed with 0.5 ml MS2/TSG-6Sepharose and 0.5 ml TTBS, and the mixture incubated in a cryotube at 4°C. overnight under constant rotation. The suspended solid phase matrixmaterial was transferred to a centrifuge tube and washed with 10 mlTTBS. Thereafter, the sediment was transferred to an Eppendorf tube,centrifuged (14,000 rpm, 2 min.) and the supernatant carefully removed.One ml 0.1M glycine-HCl buffer, pH 2.5, was added and the gel wasvigorously shaken for 2 min. After further centrifugation, thesupernatant was immediately neutralized with solid Tris.

EXAMPLE X Detection of Natural and Recombinant TSG-6 Protein fromTNF-Treated FS-4 Cells and GM-637 Cells Transfected with a TSG-6Expression Vector

[0286] The predicted TSG-6 protein sequence features a putativeinitiation methionine, followed by eleven hydrophobic amino acids and acharged region. This portion has the characteristics of a cleavablesignal peptide. There are proteins that are known to exist in bothsecreted and membrane-anchored form (either integral orphosphatidylinositol-linked) (Mosley, B. et al, Cell 59:335 (1989);Camerini, D. et al, Nature 342:78 (1989)). Thus, the TSG-6 protein mayalso be anchored to the cell surface via glycosyl-phosphatidylinositolstructures because of the hydrophobic amino acid stretches in itsC-terminal portion which could interact with the cell membrane. Todistinguish between these possibilities, experiments were conducted tolocalize TSG-6 protein in the supernatants or extracts of either (a)FS-4 cells treated with TNF for 7-24 hours, or (b) GM-637 cellstransfected with TSG-6 cDNA (termed GSV-L5).

[0287] After FS-4 cells had been grown to confluence in MEM containing5% FCS, the medium was exchanged for MEM containing 0.25% FCS and thecells maintained in this medium for three days. Thereafter, the mediumwas removed and the FS-4 cells received MEM containing 0.25% FCS with orwithout 20 ng/ml TNF-α. After 5 h the medium was exchanged forserum-free MEM containing nonessential amino acids. (TNF-treatedcultures again received 20 ng/ml TNF-α). GSV-L5 cells were grown toconfluence in MEM containing 10% FCS and 800 μg/ml G418. Thereafter, themedium was replaced with serum-free MEM containing nonessential aminoacids. Both FS-4 and GSV-L5 cells were cultured for a total of 7 to 24hours, after which culture supernatants and cell pellets were collectedand processed. Cell culture supernatants were collected, cleared bycentrifugation, and concentrated about 100-fold in an Amicon apparatus.Cell pellets were washed with serum-free medium and lysed in SDS-PAGEsample buffer.

[0288] For Western blot analysis of samples 12.5% polyacrylamide gelswere used in a Mini-Protean II Electrophoresis cell (Bio-Rad). Theelectrophoretic transfer was carried out at 100V for 1 hr usingnitrocellulose as transfer medium (Trans-Blot Transfer Medium, Bio-Rad).After blocking with 1% “blotto” in Tris buffered saline,affinity-purified rabbit anti-TSG-6 antiserum was used as the firstantibody, a biotinylated goat anti-rabbit immunoglobulin was used as thesecond antibody, and an avidin-biotinylated alkaline phosphatase complex(Vectastain, Vector Labs.) was used as the detection system.

[0289] The immunopurified antibody, specific for TSG-6 domains of theTrpE/TSG-6 fusion protein, detected one or more bands in concentratedsupernatants of TNF-treated FS-4 cells or GSV-L5 cells, but not insupernatants from control cells (FIG. 13A). These bands were notdetected by an immunopurified pre-immune serum from the same rabbit(FIG. 13B). No bands could be detected by the immunopurified antibody inlysates of GSV-L5 cells (FIG. 14) or lysates of FS-4 cells. The majorband detected in serum-free culture supernatants of TNF-treated FS-4cells and GSV-L5 cells (FIG. 13A) corresponds to a molecular weight of38 to 41 kDa and is thought to represent a glycosylated monomer of theTSG-6 protein. Bands corresponding to a molecular weight of greater than110 kDa were sometimes found in serum-free culture supernatants ofGSV-L5 cells (FIG. 13A) and FS-4 cells, probably representing anoligomeric or glycosaminoglycan-linked form of the TSG-6 protein.Another band, corresponding to a molecular weight of approximately 32 kDand probably representing the nonglycosylated or partially glycosylatedTSG-6 monomer, could be found in affinity-purified TSG-6 preparationsfrom serum-containing GSV-L5 cultures (FIG. 16) and sometimes inconcentrated supernatants of serum-free GSV-L5 cultures (FIG. 15).

EXAMPLE XI Binding of TSG-6 Protein to Hyaluronic Acid

[0290] To analyze the hyaluronic acid binding properties of the TSG-6protein, hyaluronic acid was coupled to Sepharose (HA-Sepharose) to beused as a matrix for affinity chromatography.

[0291] It is known that the binding of the cartilage proteoglycan coreprotein and link protein to hyaluronic acid, though highly specific, ismaintained at least partially by ionic interactions. Basic amino acidresidues in the highly conserved hyaluronic acid binding domain of theproteoglycan core protein (Hardingham et al, Biochem. J. 157:127 (1976))and in the link protein (Lyon, M., Biochim. Biophys. Acta 881:22 (1986))are essential for this interaction. Modification of the uronic acidresidues in hyaluronic acid also interferes with the protein-hyaluronicacid interaction (Christner, J. et al, Biochem. J. 167:711 (1977)). Highsalt concentrations prevent the binding of link protein to hyaluronicacid and dissolve link protein-hyaluronic acid complexes (Goetinck, P.F. et al, J. Cell Biol. 105:2403 (1987); Tengblad, A., Biochem. J.199:297 (1981)).

[0292] Because the TSG-6 protein shows considerable homology to thehyaluronic acid binding domains of proteoglycan core protein, high saltconcentrations were used for elution in experiments designed to showbinding of TSG-6 protein to HA-Sepharose.

[0293] To couple hyaluronic acid to Sepharose, 100 mg hyaluronic acidfrom bovine trachea (Sigma Chemicals) was dialyzed extensively against0.5M NaCl. Five ml EAH-Sepharose 4B (Pharmacia) was washed extensivelywith 0.5M NaCl and mixed with the hyaluronic acid solution. The pH wasadjusted to 4.5 and 40 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (Aldrich Chem. Co.), dissolved in 1 ml distilled water,was added dropwise under constant stirring. The pH was kept at 4.5 for 1hr. After constantly stirring the reaction mixture overnight, 200 μlacetic acid were added for another 4 hr to block the remaining aminogroups on the EAH-Sepharose. Finally, the matrix material was washedseveral times, alternating between 0.1M acetate buffer (pH 4.0,containing 0.5 M NaCl) and 0.1M Tris-HCl buffer (pH 9.5, containing 0.5M NaCl), and was resuspended in phosphate buffered saline. ControlSepharose was prepared in the same way but without hyaluronic acid.

[0294] To show the binding of TSG-6 protein to HA-Sepharose but not to asimilarly activated and blocked control Sepharose matrix, concentratedserum-free supernatant of GSV-L5 cells was used. HA-Sepharose or controlSepharose (200 μl each) was incubated with 500 μl concentratedsupernatant of GSV-L5 cells and 500 μl phosphate buffered saline (PBS)overnight at 4° C. under constant rotation. Thereafter, the supernatantswere removed and analyzed by Western blotting with anti-TSG-6 antibody.The Sepharose was washed with 10 ml PBS and 10 ml PBS containing 0.05%Tween-20. Thereafter, the HA-Sepharose as well as the control Sepharosewere eluted with 1 ml 20 mM Tris-HCl, pH 8.5, containing 3M NaCl. Theprocedure was carried out in Eppendorf tubes. After vigorous shaking andcentrifugation the supernatants were removed, dialyzed against Trisbuffered saline and analyzed by Western blotting (FIG. 15). Whereas thecontrol Sepharose matrix did not bind any detectable TSG-6 protein, theHA-Sepharose bound virtually all the TSG-6 protein present in theconcentrated GSV-L5 culture supernatant. The band showing a molecularweight of about 29 or 32 kDa, detectable in supernatants from GSV-L5cells after prolonged cultivation (20-24 hr) in serum-free medium,probably represents a nonglycosylated or partially glycosylated TSG-6monomer.

[0295] When supernatants from GSV-L5 cultures in serum-containing medium(10% FCS) were used for the affinity chromatography on HA-Sepharose,three distinct bands could be detected in Western blots (FIG. 16). Oneband corresponding to a molecular weight of about 29 or 32 kDa probablyrepresented a nonglycosylated or partially glycosylated TSG-6 monomer.The main band corresponding to a molecular weight of 36 or 38 to 41 kDaprobably represented an N-glycosylated monomer. A third (and morediffuse) band corresponding to a molecular weight of greater than 110kDa was thought to represent a TSG-6 oligomer or aglycosaminoglycan-linked form of the TSG-6 protein.

EXAMPLE XII TSG-6 Protein and Leukocyte Adhesion

[0296] Based on homology with CD44/Hermes, an important lymphocytehoming receptor, TSG-6 is expected to play a role in leukocyte adhesion.This was tested by treating FS-4 cells with TNF and showing that thisled to a marked increase in the adherence of human “PHA blasts”(peripheral blood T cells cultured in the presence of phytohemagglutinin(PHA) and IL-2). TNF increases lymphocyte adhesion to endothelial cells,mediated at least in part by the up-regulation of the adhesion molecule,ICAM-1 (Dustin, M. L. et al, J. Immunol. 137: 245 (1986)). TNF alsoup-regulates the neutrophil adhesion molecule, ELAM-1, in HUVEC(Bevilacqua, M. P. et al, Proc. Natl. Acad. Sci. USA 84: 9238 (1987);Science 243:1160 (1989)).

[0297] Quantitation of lymphocyte (e.g., PHA blast) and neutrophiladherence is performed essentially according to published methods(Dustin et al, supra; Bevilacqua et al, supra)

[0298] Antibodies specific for the TSG-6 protein, as described herein,are examined for their ability to inhibit the increase in the adherenceof PHA blasts to TNF-treated FS-4 cells. Treatment of FS-4 cells withrabbit anti TSG-6 antibodies, and in particular, polyclonal antibodiesand mAb which are specific for a TSG-6 epitope which is homologous toCD44/Hermes, at the time of TNF induction, are found to reduce T celladhesion.

[0299] Specificity of this interaction is shown using mAbs specific forCD44/Hermes (such as those produced by Dr. Eugene Butcher (StanfordUniversity) which do not bind to TSG-6 (Jalkanen, S. et al, J. CellBiol. 105:983 (1987)). The anti-CD44/Hermes antibodies do not block theabove adhesion reaction, indicating that it is due to TSG-6 and notCD44/Hermes expression in TNF-treated FS-4 cells.

[0300] The role of ICAM-1 in the adherence of TNF-treated FS-4 cells,via the induction of TSG-6, is analyzed using mAbs to ICAM-1 and/or toits ligand LFA-1(Dustin et al, supra). Antibodies to ICAM-1 and LFA-1are found to reduce PHA blast adherence to TNF-treated FS-4 cells at thelevel of the T cells. Antibodies to TSG-6, and peptides of TSG-6corresponding to the portion homologous to CD44) are also found toinhibit PHA blast adhesion to TNF-treated FS-4 cells, at the level ofthe FS-4 cell.

[0301] The importance of TSG-6 in neutrophil adhesion to FS-4 cells andthe role of ELAM-1 (Bevilacqua et al, supra) is evaluated usinganti-TSG-6 antibodies, as above, and mAbs specific for ELAM-1. It isshown that both types of antibodies inhibit neutrophil adherence toTNF-treated FS-4 cells, indicating an interaction between ELAM-1 onneutrophils and TSG-6 (or a TSG-6-dependent process) in fibroblasts.

EXAMPLE XIII Inhibition of TNF-induced, TGF-6-mediated Release ofProteoglycan from Cartilage Explants by Anti-TSG-6 Antibodies

[0302] Pieces of articular cartilage (approximately 4 mg wet weight)obtained from patients undergoing surgery or biopsy are maintained for48 h at 37° C. in DMEM containing 10% FCS. Each piece is thentransferred to a well of a 96-well plate and incubated in 0.2 ml ofculture medium, either with no addition, with human TNF, or with humanTNF and an anti-TSG-6 antibody. The medium is changed at about 3 daysand the culture is terminated after about 6 days. The cartilage explantis removed from the medium and digested completely with papain. Thechondroitin sulfate content of this digest and of the culture medium isestimated by use of the metachromatic dye, dimethylmethylene blue(Oldberg, A. et al, J. Biol. Chem. 256: 10847 (1981)). TNF is found toinduce release of proteoglycan from cartilage and antibodies to TSG-6inhibit this breakdown, indicating that TSG-6 is a mediator ofTNF-induced proteoglycan release.

EXAMPLE XIV

[0303] Experimental Procedures

[0304] Materials Chondroitin sulfate ABC lyase from Proteus vulgaris (EC4.2.2.4) and hyaluronate lyase from Streptomyces hyalurolyticus (EC4.2.2.1) were purchased from Sigma, rabbit anti-human inter-α-inhibitor(IαI) immunoglobulin was from Dako (Glostrup, Denmark) and[³⁵S]methionine/[³⁵S]cysteine (Trans ³⁵S-label) was purchased from ICN.Immunoprecipitin (heat-killed, formalin fixed Staphylococcus aureas,SAC) was from GIBCO BRL, and EX-CELL 300 and EX-CELL 400 medium was fromJRH Biosciences (Lenexa, Kans.). Albumin Removal AFFINI-FILTERS werefrom Affinity Technology, New Brunswick, N.J., Centricon-10concentrators were from Amicon, and polyvinylidene difluoride (PVDF)membranes (Immobilon-P) were from Millipore. FPLC equipment andseparation matrices were from Pharmacia. All other chemicals werepurchased from commercial suppliers and were of analytical or molecularbiology grade.

[0305] Production and Purification of Recombinant Human TSG-6 ProteinRecombinant Autographa californica nuclear polyhedrosis virus (genusBaculovirus) containing human TSG-6 cDNA (Wisniewski et al, Physiologyand Pathophysiology of Cytokines (1992)) was used for the infection ofHigh Five insect cells from Trichoplusia ni (BTI-TN-5B1-4; purchasedfrom Invitrogen, San Diego, Calif.). For high-level expression andpurification of TSG-6 protein, TN-5B insect cells were grown inserum-free EX-CELL 400 medium. The cell culture medium was replaced 24hrs. after inoculation of ˜70-80% confluent cultures with recombinantvirus and collected again 48 hrs. later. For purification of TSG-6protein, cleared culture supernatant was directly loaded on a MonoS(HR5/5) column equilibrated with 20 mM 4-morpholineethanesulfonic acid(MES), pH 6.5. Bound protein was eluted with a linear gradient (20 mL)from 0 to 1 M NaCl in 20 mM MES, pH 6.5. Fractions containing the bulkof TSG-6 protein (0.45-0.65 M NaCl) were pooled, concentrated inCentricon-10 units, and applied onto a Supperdex 75 (HR 10/30) columnequilibrated with 20 mM MES, pH 6.5, 0.5 M NaCl. This resulted in therecovery of ≧95% pure TSG-6 protein as judged by silver staining ofSDS-PAGE gels. About 1 μg of pure protein was recovered per 1 mL ofculture supernatant. Microsequencing of the purified TSG-6 proteinestablished Trp¹⁸ as the N-terminus of the mature secreted glycoprotein(Table 2), which is in good agreement with the predicted cleavage siteof the signal peptide sequence (Von Heijne, J. Mol. Biol. 173:243-251(1984); J. Mol. Biol 184:99-105 (19850).

[0306] Analysis of TSG-6 Binding to Carrier Protein To determine thepresence of TSG-6 binding protein, samples to be analyzed were mixedwith an equal volume of TSG-6-containing serum-free supernatants ofTN-5B insect cells infected with recombinant Baculovirus and incubatedat 37° C. for 30-60 min. (Purified TSG-6 protein was used in someexperiments, as indicated.) Thereafter, the mixtures were analyzed forthe presence of the 120-kDa complex by Western blotting with purifiedrabbit antibody to TSG-6 as described earlier (Wisniewski et al, J.Immunol. 151:6593-6601 (1993)). For detection of inter-α-inhibitor (IαI)epitopes by Western blotting, membranes were incubated with a rabbitanti-human IαI antibody (Dako) at a 1:2000 dilution for 1 hr.

[0307] Immunoprecipitation Human HepG2 hepatoma cells producing IαIconstitutively (Bourguignon et al, Biochem. J. 261:305-308 (1989)) weregrown in serum-free EX-CELL medium. [³⁵S]Methionine(Trans³⁵S-label) wasadded to about 75% confluent HepG2 cell cultures in a 25 cm² flask (500μCi/culture), and the culture supernatant was collected after 24 hrs.The ³⁵S-labeled HepG2 culture supernatant (300 μL) was incubated with 16μg of purified recombinant TSG-6 protein or with buffer for 1 hr. at 37°C. Samples were precleared with 150 μL of 10% SAC. Rabbit anti-TSG-6antiserum or preimmune serum from the same rabbit (5 μL) was added tothe supernatants and incubated for 3 hrs. at 37° C. Thereafter, 150 μLof 10% SAC was added and incubated for 30 min. at room temperature. Thesupernatants were removed, and the pellets were washed 3 times with 1 mLof 20 mM tris, pH 7.5, 0.5 M NaCl, and 0.02% Tween-20. Pellets wereresuspended in 40 μL of SDS-PAGE buffer (reducing) and incubated for 3min. in a boiling water bath. The supernatants were removed and analyzedby SDS-PAGE on 10% polyacrylamide (PAA) gels and fluorography.

[0308] Partial Purification of TSG-6 Binding Protein from Human Serumfor N-Terminal Microsequencing Protein precipitated between 40% and 55%saturation with ammonium sulfate from 40 mL of fresh human serum wasdissolved in 10 mL of PBS and dialyzed against 50 mM KH₂PO₄, pH 7.0,5-mM NaCl. Four aliquots were passed through Affini-filter cartridgesfor albumin removal (Affinity Technology). Each cartridge was washedwith 5 mL of the same buffer and eluted with 5 mL of 5 mM KH₂PO₄, pH7.0, 0.5 M NaCl. The eluates of four cartridges were pooled, dialyzedagainst 20 mM Tris, pH 7.5, 50 mM NaCl, and loaded on a MonoQ column(HR5/5, Pharmacia) using a Pharmacia FPLC system. Protein was elutedwith a linear gradient (16 mL) from 50 mM to 1 M NaCl in 50 mM Tris, pH7.5. Fractions containing TSG-6 binding activity were pooled andconcentrated in Centricon-10 units to a final volume of 200 μL. Thismaterial was further separated by FPLA on a Superdex 200 column (HR10.30). The column was equilibrated with 50 mM Na₂HPO₄, pH 7.0, 150 mMNaCl and run at a flow rate of 0.5 mL/min. Fractions containing TSG-6binding activity were pooled and concentrated in Centricon-10 units. Thematerial was further separated by SDS-PAGE under reducing conditions ona 4-15% PAA gel and transferred in methanol-free transfer budder at 200mA for 1 hr. to a PVDF membrane. Staining with Coomassie Blue R250revealed only one protein band greater than 200 kDa which was used formicrosequencing.

[0309] Purification of IαI from Human Serum IαI purified from humanserum, according to Salier et al, Anal Biochem. 109:273-283 (1980), withsome modifications. FPLC on Q Sepharose Fast Flow was used instead ofDEAE-Sephacel chromatography. Chelating Sepharose Fast Flow was used forzinc chelate chromatography. Phenyl Superose was used for Hydrophobicchromatography and Superdex 200 was used for size-exclusionchromatography instead of Sephacryl-300. The IαI recovered wasessentially pure as judged by SDS-PAGE and silver staining.

[0310] Protein Sequencing Coomassie Blue-stained protein bands on thePVDF membranes ere cut from the blots and placed directly into amicro-cartridge on an Applied Biosystems Model 473A protein sequencer.Automated Edman degradations were performed using standard cycles withgas phase delivery of trifluoroacetic acid (TFA). Data collection andreduction were performed using Applied Biosystems Model 610 software.

[0311] Microsequencing of the TSG-6/IαI Complex Purified recombinantTSG-6 (2.5 μg) was incubated with 2.6 μg of IαI purified from humanserum for 1 hr. at 37° C. After SDS-PAGE in an 8% PAA gel under reducingconditions, protein was transferred to a PVDF membrane in methanol-freetransfer buffer at 200 mA for 1 hr. The membrane was stained withCoomassie Blue R250, and the newly formed 120-kDa band (not present inthe IαI or TSG-6 preparation) was excised for microsequencing.

[0312] Results

[0313] Binding of TSG-6 Protein to a Protein Present in Mammalian Seraand in Supernatants of Human HepG2 Hepatoma Cells Western blot analysisof serum-free culture supernatant of TN-5B insect cells infected withrecombinant Baculovirus encoding human TSG-6 revealed the presence of a32-kDa band reactive with antibody to TSG-6 (FIG. 18, lane 1).Recombinant human TSG-6 protein produced in insect cells migratessomewhat faster than the 35-kDa TSG-6 protein from human cells(Wisniewski et al, J. Immunol. 151:6593-6601 (1993)) possibly due to sdifferent extent of glycosylation. A second band recognized by antibodyagainst TSG-6, with the apparent molecular mass of 29 kDa, probablyrepresent unglycosylated TSG-6 protein. When TSG-6 containing culturesupernatants were incubate at 37° C. in the presence of fetal bovineserum (lane 3), mouse serum (lane 7), or serum-free culture supernatantof human HepG2 hepatoma cells (lane 5), an additional 120-kDa bandbecame readily apparent. A band of identical electrophoretic mobilityappeared after incubation of recombinant TSG-6 protein with human orrabbit serum.

[0314] The 120-kDa Band Represents a Complex of TSG-6 with a DistinctProtein In order to show that the newly formed 120-kDa band revealed byWestern blot analysis is indeed a complex of TSG-6 with a distinctprotein, and not a TSG-6 oligomer whose formation is promoted by serum,we employed immunoprecipitation. When a supernatant from HepG2 cellscultured in serum-free medium in the presence of [³⁵S]methionine wasincubated with unlabeled purified recombinant TSG-6 protein andimmunoprecipitated with a rabbit anti-serum to TSG-6, a labeled 120-kJan. 9, 2001 a molecule was precipitated (FIG. 19). Thisimmunoprecipitation was specific because incubation with preimmune serumfrom the same rabbit or incubation of ³⁵S-labeled HepG2 supernatantswith immune serum in the absence of TSG-6 protein failed to precipitatea labeled 120-kDa molecule. [³⁵S]Methionine incorporation into amolecule specifically recognized by an antibody to TSG-6 indicates thatthe 120-kDa molecule is indeed a complex of TSG-6 protein with anotherdistinct protein that is constitutively produced and secreted by humanHepG2 cells.

[0315] Partial Purification and Identification of the TSG-6 BindingProtein from Human Serum Fractionation of normal human serum by ammoniumsulfate precipitation showed that proteins precipitated between 40% and55% saturation contained most of the TSG-6 binding activity, althoughsignificant binding was also detected in the fraction precipitated at anammonium sulfate saturation of 40%. The purification procedure used forthe isolation of the binding protein and the Western blot-based assayused for the detection of TSG-6 binding protein are described underExperimental Procedures. During Affini-filter chromatography. Most ofthe TSG-6 binding activity eluted at 0.5 M NaCl together with residualalbumin. FPLC on a MonoQ column proved to be very efficient for furtherpurification of the TSG-6 binding protein. SDS-PAGE followed by silverstaining revealed that incubation of fractions from MonoQ-FPLC withrecombinant TSG-6 protein resulted in the partial disappearance ofa >200-kDa band and the appearance of a new band at 120 kDa (FIG. 20).This finding suggested that the human TSG-6 binding protein is greaterthan 200 kDa in size and hence considerably greater than its complexwith TSG-6. During FPLC on Superdex 200, the TSG-6 binding proteineluted with a retention volume corresponding to a molecular mass ofabout 270 kDa. Fractions containing TSG-6 binding activity wereconcentrated about 80-fold before SDS-PAGE was performed on 4-15% PAAgradient gels or 8% PAA gels under reducing conditions. Proteins weretransferred electrophoretically to PVDF membranes. CBB staining revealedthe presence of only one band greater than 200 kDa which was cut formicrosequencing.

[0316] Microsequencing of the TSG-6 binding protein resulted in doublesignals for the first five cycles and one signal for each of thefollowing seven cycles. Comparison of the resulting sequences withsequence stored in protein databases revealed that they identity 2 ofthe 3 chains of the human IαI (trypsin): the bikunin chain of IαI waspresented by its 12 N-terminal amino acids, whereas the heavy chain 2(HC2) was represented by its 5 N-terminal amino acids (Table 1). It isnoteworthy that Ser¹⁰ of the bikunin chain could not be identified. Nosignals corresponding to the heavy chain 1 (HC1) of IαI were retrieved.The microsequencing data along with the molecular mass of ˜250 kDa(determined by SDS-PAGE) indicate that the TSG-6 binding protein is IαIrather than inter-α-like-inhibitor (IαLI) whose molecular mass is130-140 kDa (Enghild et al, J. Biol. Chem. 264:15975-15981 (1989); Rouetet al, Biol. Chem. Hoppe-Seyler 373:1019-1024 (1992)). Subsequentmicrosequencing of another preparation of IαI purified from human serumallowed the identification of HC1 besides HC2 and bikunin. However, thedetected amount of HC1 was substantially lower than that of the twoother chains on a molar basis. TABLE 1 N-Terminal Amino Acid Sequence ofthe Human TSG-6 Binding Protein Determined by Microsequencing PositionAmino Acid Residues  1 Ala Ser  2 Val Leu  3 Leu Pro  4 Pro Glu  5 GlnGly  6 Glu —^(a)  7 Glu —  8 Glu —  9 Gly — 10 Xaa — 11 Gly — 12 Gly —(SEQ ID NO: 7) (SEQ ID NO: 8)

[0317] Rapid Complex Formation between Recombinant TSG-6 Protein and IαIPurified from Human Serum at 37° C. The 120-kDa TSG-6/IαI complex formedreadily when purified TSG-6 protein and purified IαI were incubatedtogether at 37° C. but not at 0° C. (FIG. 21. At 37° C., complexformation was detectable within 2 min., and the reaction appeared to becomplete by 10 min. At 0° C., however, little or no TSG-6/IαI complexwas formed within 1 hr. (FIG. 21). Mono-specific rabbit antisera againstwith TSG-6 protein or IαI (FIG. 22) detect the 120kDa complex in Westernblots, indicating the presence of both TSG-6 and IαI epitopes in astable complex. Besides the formation of the TSG-6/IαI complex of 120kDa, incubation of TSG-6 protein with IαI resulted in the appearance ofyet another band with a molecular mass of ˜kDa (FIG. 23, line 2) whichwas detected by anti IαI but not by anti-TSG-6. This IαI derivativeappears to be a byproduct of the reaction of TSG-6 with IαI. It shouldbe noted that the 120kDa TSG-6/IαI complex is formed by purified TSG-6and IαI proteins in the apparent absence of other proteins.

[0318] Composition of the TSG-6/IαI Complex The 120-kDa complex, formedby incubating together purified TSG-6 protein and IαI, was isolated andidentified by SDS-PAGE in an 8% PAA gel, electrotransfer to a PVDFmembrane, and CBB staining. Microsequencing of the isolated bandcorresponding to the complex revealed the presence of TSG-6 (shown belowin Table 2 as an amino acid sequence corresponding to amino acids 18-27of SEQ ID NO:2) protein (shown below in Table 2 as an amino acidsequence corresponding to amino acids 10-27 of SEQ ID NO:2), bikunin,and HC2 of IαI in nearly equimolar ratios (Table 2). Interestingly,Ser¹⁰ of the bikunin chain, which was not detectable duringmicrosequencing of IαI (see Table 1), was found in an equimolar amountin the TSG-6/IαI complex. The unexpected appearance of a serine residuein position 10 could reflect the presence of an additional chain orpartial modification of one or more N-termini. A less likely possibilityis that Ser¹⁰ of the bikunin chain, which is glycosylated in IαI, mightbecome deglycosylated in the process of TSG-6/IαI complex formation.TABLE 2 N-terminal Amino Acid Sequences of the TSG-6/IαI ComplexDetermined by Microsequencing Yield (pmol of Amino Acid Residuesphenylthiohydantoin Position Bikunin HC2 TSG-6 (PTH)) 1 Ala Ser Trp 1012 15 2 Val Leu Gly 10 12 12 3 Leu Pro Phe  9 11  9 4 Pro Gly Lys  9 15 8 5 Gln Glu Asp  7 12 13 6 Glu Ser Gly 11  8 13 7 Glu Glu Ile 13  9 8Glu Glu Phe 15  9 9 Gly Met His 11  9  6 10  Ser Met Asn  8 12 14 SEQ IDSEQ ID SEQ ID NO:9 NO:10 NO:2 (18-27

[0319] The Stable Crosslink in the TSG-6/IαI Complex is Provided by aGylcosaminoglycan Chain The stability of the 120 kDa TSG-6/IαI complexduring SDS-PAGE under reducing conditions raises the question of thenature of the interaction between TSG-6 and the other components of thecomplex. Addition of 8 M urea before SDS-PAGE did not affect the 120-kDacomplex (FIG. 23). The effect of treatment with 8 M guanidinehydrochloride could not be fully evaluated because the detectability ofboth the TSG-6 band and the TSG-6/IαI Western blots was significantlydiminished. However, the TSG-6/IαI complex was still detectable aftertreatment with 8 M guanidine hydrochloride. Taken together, these datareflect the high stability of the TSG-6/IαI complex, and the formationof a covalent bond cannot be ruled out.

[0320] It is known that the three polypeptide chains of IαI arecross-linked by a chondroitin sulfate chain (Enghild et al, J. Biol.Chem. 264:15975-15981 (1989); Jenssen et al, FEBS Lett. 230:195-200(1988); Balduyck et al, Biol. Chem. Hoppe-Seyler. 370:329-336 (1989). Ithas been shown that bikunin, HC2 of IαLI, and HC3 of pre-α-inhibitor(PαI) are covalently bound to chondroitin 4-sulfate (Enghild et al, J.Biol. Chem. 264:15975-15981 (1989); J. Bio. Chem. 266:747-751 (1991); J.Biol. Chem. 268:8711-8716 (1993). To determine if cross-linking viachondroitin sulfate is also required for the stability of the TSG-6/IαIcomplex, purified TSG-6 protein and IαI were first incubated together toallow the formation of the complex and then treated with chondroitinsulfate ABC lyase from Proteus vulgaris or hyaluronidase fromStreptomyces hyalurolyticus. Treatment with chondroitin sulfate ABClyase resulted in complete disappearance of the complex whereashyaluronidase has no effect (FIG. 24).

[0321] Chondroitin Sulfate Associated with IαI is Required for theFormation of the TSG-6/IαI Complex In order to determine the effect ofchondroitin sulfate ABC lyase on either TSG-6 protein or IαI separately,purified TSG-6 protein and purified IαI from human serum were incubatedwith a limited amount of chondroitinase for 16 hrs. and then mixed withuntreated IαI or TSG-6 protein, respectively. Western blotting revealedthat IαI preincubated with chondroitin sulfate ABC lyase was no longerable to form a complex with untreated TSG-6 protein (FIG. 25). On theother hand, pretreatment with chondroitinase had little effect on theability of TSG-6 protein to react with untreated IαI (FIG. 25). A slightdecrease of the amount of TSG-6/IαI complex formed bychondrioitinase-pretreated TSG-6 (FIG. 25) can be explained bycarry-over of the enzyme into the final TSG-6-IαI incubation mixturebecause the chondroitinase could not be selectively inactivated.Limiting chondroitinase activity to the necessary minimum was essentialin this experiment in order to prevent IαI inactivation during the finalincubation. This finding suggests that IαI, but not TSG-6 protein, has achondroitinase-sensitive structure required for the formation of theTSG-6-IαI complex.

[0322] Discussion

[0323] We showed earlier that TSG-6 synthesis is rapidly induced inhuman diploid fibroblasts and peripheral blood mononuclear cells afterstimulation with the inflammatory cytokines TNF and IL-1 (Lee et al,Mol. Cell. Biol. 10:1982-1988 (1990); J. Biol. Chem. 268:6154-6160(1993); Wisniewski et al, J. Immunol. 151:6593-6601 (1993)). Theaffinity of TSG-6 protein for hyaluronan suggests a possible associationwith the extracellular matrix and cartilage (Lee et al, J. Cell. Biol.116:545-557 (1992)). High levels of TSG-6 protein in the synovial fluidof patents with rheumatoid arthritis and constitutive TSG-6 expressionby cultured synovial cells from rheumatiod joints in vitro that wasfurther enhanced by TNF and IL-1, suggested a role for TSG-6 ininflammatory diseases of connective tissue and cartilage (Wisniewski etal, J. Immunol. 151, 6593-6601 (1993)). Little is known, however, aboutthe actions of TSG-6 at the molecular or cellular level. Here we showthat recombinant human TSG-6 protein is readily incorporated into astable 120 kDa complex if incubated with human, fetal bovine, rabbit, ormouse serum. Isolation and microsequencing of the human TSG-6 bindingprotein allowed its identification as IαI, an extensively studied serumprotein.

[0324] IαI is a complex protein in which the bikunin chain is linked toHC1 and HC2 (Enghild et al, J. Biol. Chem. 264:15975-15981 (1989);Gebhard et al, Biol. Chem. Hoppe-Seyler 371 Suppl.:13-22 (1990); Rouetet al, Biol. Chem. Hoppe-Seyler 373:1019-1024 (1992)). Yet, sequenceanalysis of the TSG-6 binding protein purified from human serum revealedonly the bikunin chain and HC2 (Table 1), and no indication for thepresence of HC1 was obtained. Subsequent sequencing of anotherpreparation of IαI purified from human serum resulted in the detectionof very weak signals for the sequence of HC1, corresponding to aboutone-tenth of the other two chains on a molar basis. Other investigatorswho reported N-terminal sequencing data for IαI also received incompleteand divergent sequences for HC1 when sequencing the unmodified IαImolecule (Enghild et al, J. Biol. Chem. 264:15975-15981 (1989); Jessenet al, FEBS Lett. 320:195-200 (1988); Malki et al, Biol. Chem.Hoppe-Seyler 373:1009-1018 (1992)). Nevertheless, little doubt existsthat the TSG-6 binding proteins for IαI forms what appears to be thesame 120 kDa complex with TSG-6 proteins whole serum (FIGS. 21 and 22).While there is no doubt that IαI is the binding TSG-6 protein we haveisolated, it is possible that IαLI or PαI also can bind TSG-6 protein.

[0325] Evidence that the reaction between TSG-6 and IαI indeed yields acomplex of TSG-6 with one or more polypeptide chains of IαI is providedby the immunoprecipitation data (FIG. 19). Further information about thecomposition of the TSG-6/IαI complex is derived from Western blotanalysis. Antisera specific for either TSG-6 protein or IαI detected a120 kDa band newly formed upon incubation of TSG-6 protein and IαI witheach other (FIG. 22), suggesting the presence of TSG-6 and IαI epitopesin the complex. This was confirmed by microsequencing of the TSG-6/IαIcomplex (Table 2) which revealed the presence of three chains: TSG-6protein, bikunin, and HC2. The signals of all three chains arerepresented in nearly equimolar amounts, suggesting that the complexcontains one of each polypeptide chain. No signals corresponding to HC1could be detected. The molecular mass of the complex is surprisingly lowif one considers the molecular masses of the incorporated polypeptides.HC2 has a molecular mass of 70 kDa (Enghild et al, J. Biol. Chem. 264,15975-15981 (1989)), the reported molecular mass of bikunin is 26-70 kDadepending on the extent of glycosylation (Gebhard et al, Biol. Chem.Hoppe-Seyler 371 Suppl:13-22 (1990); Rouet et al, Biol. Chem.Hoppe-Seyler 373:1019-1024 (1992)), and that of recombinant TSG-6protein is 32 kDa. The fact that the apparent molecular mass of the 120kDa complex is less than the sum of its components suggests that someadditional modifications, such as deglycosylation or limited proteolyticcleavage, might take place. Alternatively, changes of the grossstructure of the complex due to incorporation of TSG-6 could have asubstantial effect on its apparent molecular mass.

[0326] The unusual stability of the TSG-6/IαI complex raises thequestion of the nature of the bonds linking its components. Theresistance of the complex to boiling in 2% SDS and 5% β-mercaptoethanolas well as to 8 M urea makes any noncovalent hydrophobic or hydrophilicbond unlikely. In addition, the strict temperature dependence ofTSG-6/IαI complex formation (FIG. 22) suggests that the reactioninvolves an activated transition state and supports the notion that acovalent bond is formed. However, an unusually stable noncovalentassociation of TSG-6 with the glycosaminoglycan chain of the complexcannot be ruled out. The polypeptide chains of IαI are cross-linked bychondroitin 4-sulfate chain (Enghild et al, J. Biol. Chem. 264,15975-15981 (1989); Jessen et al, FEBS Lett. 320, 195-200 (1988);Balduyck et al, Biol. Chem. Hoppe-Seyler 370, 329-336 (1989)). Thesensitivity of the TSG-6/IαI complex to chondroitin sulfate ABC lyase(FIG. 24) and the inability of chondroitinase-pretreated IαI to form thecomplex (FIG. 25) suggests that the chondroitin 4-sulfate chaincross-linking the polypeptide chains of IαI is also required for theformation of the 120 kDa complex. It has been shown that the chondroitin4-sulfate chain of IαI is bound to Ser¹⁰ of bikunin via a commonGal-Gal-Xyl oligosaccharide (Enghild et al, J. Biol. Chem.264:15975-15981 (1989); J. Biol. Chem. 266:747-751 (1991); Chirat et al,Int. J. Biochem. 23:1201-1203 (1991)). An unusual ester bond has beenshown to cross-link the α-carboxylic a group of the HC2 C-terminalAsp⁶⁴⁸ of IαLI to C-6 of an internal N-acetyl-galactosamine of thechondroitin 4-sulfate chain (Enghild et al, J. Biol. Chem. 26:8711-8716(1993)). A similar bond cross-links the C-terminal Asp⁶¹⁸ of HC3 tochondroitin 4-sulfate in PαI (Enghild et al, J. Biol. Chem. 266:747-751(1991); J. Biol. Chem. 268:8711-8716 (1993)). Analysis of the cDNAs ofthe three heavy chains showed that all have the conserved consensussequence Val-Xaa-Xaa-Asp-Pro-His-Ile-Ile (SEQ ID NO:11), supposed todetermine the cleavage site for the C-terminal propeptide (Bourguignonet al, Eur. J. Biochem. 212:771-776 (1993)) after the aspartic acidresidue. This cleavage generates the free α-carboxylic group of the nowC-terminal aspartic acid residue which forms the ester bond to aninternal N-acetylgalactosamine of chondrotin 4-sulfate, two reactionsthat may be closely coupled. Interestingly, TSG-6 also features a coreof the consensus sequence Val-Xaa-Xaa-Asp-Pro²⁴⁹ (corresponding to aminoacid residues 1-5 of SEQ ID NO:11). Hence, it is conceivable that TSG-6forms a direct covalent bond to the chondroitin 4-sulfate chain of IαI.Additional studies are required to determine the molecular structuresformed and the exact nature of the interactions leading to the formationof the exceedingly stable TSG-6/IαI complex.

[0327] Although the trypsin-inhibitory activity of IαI has been knownfor a long time (Heide et al, Clin. Chem. Acta 11:82-85 (1965)), littleis known about the functions of the different members of the IαI family.However, disease-associated presence in various tissues and fluctuationsseen in the serum levels of IαI and IαI-related proteins suggest andinvolvement in pathologic processes. Daveau et al, Biochem. J.292:485-492 (1993) reported a distinct pattern of changes in serumconcentrates of the different members of the IαI family during acuteinflammation. Proteins identical with, or closely related to, thebikunin chain of IαI have been detected in stroma and the surroundingconnective tissue of malignant tumors (Yoshida et al, Cancer 64, 860-869(1989)), in brain tissue of patients with Alzheimer's disease (Yoshidaet al, Biochem. Biophys. Res. Commun. 174:1015-1021 (1991)), and inserum and urine of patients with inflammatory disease, cancer, andleukemias (Rudman et al, Cancer Res. 36:1837-1846 (1976); Franck &Pederson, Scand. J. Clin. Lab. Invest. 43:151-155 (1976); Chawla et al,J. Cell Biochem. 42:207-217 (1990)). A link between IαI and rheumatoidarthritis was suggested over 20 years ago when Becker and Sandson,Arthritis Rheum. (1971), found IαI associated with hyaluronan in thesynovial fluid of patients with rheumatoid arthritis, whereas no IαI wasdetectable in control synovial fluids. This finding was confirmed andextended to show that IαI associates in vitro with hyaluronan isolatedfrom the synovial fluid of healthy subjects (Hutadilok et al, Ann.Rheum. Dis. 47:377-385 (1988)). Huang et al, J. Biol. Chem. 268,26725-26730 (1993) showed recently that in the presence of serum the twoheavy chains of IαI become covalently associated with hyaluronan.

[0328] The possible functional implications of complex formation betweenTSG-6 and IαI are still elusive. Though TSG-6 has been detected inhumanserum it is most prominent at sites of local inflammation (Wisniewski etal, 1993; and unpublished results). Therefore, TSG-6/IαI interactionprobably occurs most readily at inflammatory sites such as the synovialtissue in rheumatoid arthritis, where both TSG-6 and IαI are found athigh concentrations together with hyaluronan. Indeed, synovial fluidsfrom arthritis patients contained both the free 35 kDa from and the 120kDa complexed form of TSG-6 protein (Wisniewski et al, J. Immunol.151:6593-6601 (1993)). Recently, the rabbit homologue of TSG-6 has beencloned and characterized as a developmentally regulated protein (Feng &Liau, J. Biol. Chem. 268:9387-9392, 21453 (1993)), supporting the ideathat TSG-6 might play a role in developmental processes. Tissueremodeling could be the common denominator of TSG-6 association withdevelopmental and inflammatory processes. The fact that human TSG-6forms complexes of equal size with IαI from different mammalian speciesindicates that the molecular structures involved in the interaction arewell conserved among mammals, perhaps reflecting selective pressure dueto functional and structural constraints.

[0329] The references cited above are all incorporated by referenceherein, whether specifically incorporated or not.

[0330] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

[0331] While this invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications. This application is intended to cover anyvariations, uses, or adaptations of the inventions following, ingeneral, the principles of the invention and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains and as may be applied tothe essential features hereinbefore set forth as follows in the scope ofthe appended claims.

1 11 1414 base pairs nucleic acid single linear cDNA Homo sapiensFibroblast FS-4 CDS 69..899 1 GAATTCGCAC TGCTCTGAGA ATTTGTGAGCAGCCCCTAAC AGGCTGTTAC TTCACTACAA 60 CTGACGAT ATG ATC ATC TTA ATT TAC TTATTT CTC TTG CTA TGG GAA GAC 110 Met Ile Ile Leu Ile Tyr Leu Phe Leu LeuLeu Trp Glu Asp 1 5 10 ACT CAA GGA TGG GGA TTC AAG GAT GGA ATT TTT CATAAC TCC ATA TGG 158 Thr Gln Gly Trp Gly Phe Lys Asp Gly Ile Phe His AsnSer Ile Trp 15 20 25 30 CTT GAA CGA GCA GCC GGT GTG TAC CAC AGA GAA GCACGG TCT GGC AAA 206 Leu Glu Arg Ala Ala Gly Val Tyr His Arg Glu Ala ArgSer Gly Lys 35 40 45 TAC AAG CTC ACC TAC GCA GAA GCT AAG GCG GTG TGT GAATTT GAA GGC 254 Tyr Lys Leu Thr Tyr Ala Glu Ala Lys Ala Val Cys Glu PheGlu Gly 50 55 60 GGC CAT CTC GCA ACT TAC AAG CAG CTA GAG GCA GCC AGA AAAATT GGA 302 Gly His Leu Ala Thr Tyr Lys Gln Leu Glu Ala Ala Arg Lys IleGly 65 70 75 TTT CAT GTC TGT GCT GCT GGA TGG ATG GCT AAG GGC AGA GTT GGATAC 350 Phe His Val Cys Ala Ala Gly Trp Met Ala Lys Gly Arg Val Gly Tyr80 85 90 CCC ATT GTG AAG CCA GGG CCC AAC TGT GGA TTT GGA AAA ACT GGC ATT398 Pro Ile Val Lys Pro Gly Pro Asn Cys Gly Phe Gly Lys Thr Gly Ile 95100 105 110 ATT GAT TAT GGA ATC CGT CTC AAT AGG AGT GAA AGA TGG GAT GCCTAT 446 Ile Asp Tyr Gly Ile Arg Leu Asn Arg Ser Glu Arg Trp Asp Ala Tyr115 120 125 TGC TAC AAC CCA CAC GCA AAG GAG TGT GGT GGC GTC TTT ACA GATCCA 494 Cys Tyr Asn Pro His Ala Lys Glu Cys Gly Gly Val Phe Thr Asp Pro130 135 140 AAG CGA ATT TTT AAA TCT CCA GGC TTC CCA AAT GAG TAC GAA GATAAC 542 Lys Arg Ile Phe Lys Ser Pro Gly Phe Pro Asn Glu Tyr Glu Asp Asn145 150 155 CAA ATC TGC TAC TGG CAC ATT AGA CTC AAG TAT GGT CAG CGT ATTCAC 590 Gln Ile Cys Tyr Trp His Ile Arg Leu Lys Tyr Gly Gln Arg Ile His160 165 170 CTG AGT TTT TTA GAT TTT GAC CTT GAA GAT GAC CCA GGT TGC TTGGCT 638 Leu Ser Phe Leu Asp Phe Asp Leu Glu Asp Asp Pro Gly Cys Leu Ala175 180 185 190 GAT TAT GTT GAA ATA TAT GAC AGT TAC GAT GAT GTC CAT GGCTTT GTG 686 Asp Tyr Val Glu Ile Tyr Asp Ser Tyr Asp Asp Val His Gly PheVal 195 200 205 GGA AGA TAC TGT GGA GAT GAG CTT CCA GAT GAC ATC ATC AGTACA GGA 734 Gly Arg Tyr Cys Gly Asp Glu Leu Pro Asp Asp Ile Ile Ser ThrGly 210 215 220 AAT GTC ATG ACC TTG AAG TTT CTA AGT GAT GCT TCA GTG ACAGCT GGA 782 Asn Val Met Thr Leu Lys Phe Leu Ser Asp Ala Ser Val Thr AlaGly 225 230 235 GGT TTC CAA ATC AAA TAT GTT GCA ATG GAT CCT GTA TCC AAATCC AGT 830 Gly Phe Gln Ile Lys Tyr Val Ala Met Asp Pro Val Ser Lys SerSer 240 245 250 CAA GGA AAA AAT ACA AGT ACT ACT TCT ACT GGA AAT AAA AACTTT TTA 878 Gln Gly Lys Asn Thr Ser Thr Thr Ser Thr Gly Asn Lys Asn PheLeu 255 260 265 270 GCT GGA AGA TTT AGC CAC TTA TAAAAAAAAA AAAGGATGATCAAAACACAC 929 Ala Gly Arg Phe Ser His Leu 275 AGTGTTTATG TTGGAATCTTTTGGAACTCC TTTGATCTCA CTGTTATTAT TAACATTTAT 989 TTATTATTTT TCTAAATGTGAAAGCAATAC ATAATTTAGG GAAAATTGGA AAATATAGGA 1049 AACTTTAAAC GAGAAAATGAAACCTCTCAT AATCCCACTG CATAGAAATA ACAAGCGTTA 1109 ACATTTTCAT ATTTTTTTCTTTCAGTCATT TTTGTATTTG TGGTATATGT ATATATGTAC 1169 CTATATGTAT TTGCATTTGAAATTTTGGAA TCCTGCTCTA TGTACAGTTT TGTATTATAC 1229 TTTTTAAATC TTGAACTTTATGAACATTTT CTGAAATCAT TGATTATTCT ACAAAAACAT 1289 GATTTTAAAC AGCTGTAAAATATTCTATGA TATGAATGTT TTATGCATTA TTTAAGCCTG 1349 TCTCTATTGT TGGAATTTCAGGTCATTTTC ATAAATATTG TTGCAATAAA TATCCTTCGG 1409 AATTC 1414 277 aminoacids amino acid linear protein 2 Met Ile Ile Leu Ile Tyr Leu Phe LeuLeu Leu Trp Glu Asp Thr Gln 1 5 10 15 Gly Trp Gly Phe Lys Asp Gly IlePhe His Asn Ser Ile Trp Leu Glu 20 25 30 Arg Ala Ala Gly Val Tyr His ArgGlu Ala Arg Ser Gly Lys Tyr Lys 35 40 45 Leu Thr Tyr Ala Glu Ala Lys AlaVal Cys Glu Phe Glu Gly Gly His 50 55 60 Leu Ala Thr Tyr Lys Gln Leu GluAla Ala Arg Lys Ile Gly Phe His 65 70 75 80 Val Cys Ala Ala Gly Trp MetAla Lys Gly Arg Val Gly Tyr Pro Ile 85 90 95 Val Lys Pro Gly Pro Asn CysGly Phe Gly Lys Thr Gly Ile Ile Asp 100 105 110 Tyr Gly Ile Arg Leu AsnArg Ser Glu Arg Trp Asp Ala Tyr Cys Tyr 115 120 125 Asn Pro His Ala LysGlu Cys Gly Gly Val Phe Thr Asp Pro Lys Arg 130 135 140 Ile Phe Lys SerPro Gly Phe Pro Asn Glu Tyr Glu Asp Asn Gln Ile 145 150 155 160 Cys TyrTrp His Ile Arg Leu Lys Tyr Gly Gln Arg Ile His Leu Ser 165 170 175 PheLeu Asp Phe Asp Leu Glu Asp Asp Pro Gly Cys Leu Ala Asp Tyr 180 185 190Val Glu Ile Tyr Asp Ser Tyr Asp Asp Val His Gly Phe Val Gly Arg 195 200205 Tyr Cys Gly Asp Glu Leu Pro Asp Asp Ile Ile Ser Thr Gly Asn Val 210215 220 Met Thr Leu Lys Phe Leu Ser Asp Ala Ser Val Thr Ala Gly Gly Phe225 230 235 240 Gln Ile Lys Tyr Val Ala Met Asp Pro Val Ser Lys Ser SerGln Gly 245 250 255 Lys Asn Thr Ser Thr Thr Ser Thr Gly Asn Lys Asn PheLeu Ala Gly 260 265 270 Arg Phe Ser His Leu 275 90 amino acids aminoacid single linear peptide 3 Gly Val Phe His Val Glu Lys Asn Gly Arg TyrSer Ile Ser Arg Thr 1 5 10 15 Glu Ala Ala Asp Ile Cys Lys Ala Phe AsnSer Thr Leu Pro Thr Met 20 25 30 Ala Gln Met Glu Lys Ala Leu Ser Ile GlyPhe Glu Thr Cys Arg Tyr 35 40 45 Gly Phe Ile Glu Gly His Val Val Ile ProArg Ile His Pro Asn Ser 50 55 60 Ile Cys Ala Ala Asn Asn Thr Gly Val TyrIle Leu Thr Ser Asn Thr 65 70 75 80 Ser Gln Tyr Asp Thr Tyr Cys Phe AsnAla 85 90 98 amino acids amino acid single linear peptide 4 Gly Val ValPhe Pro Tyr Phe Pro Arg Leu Gly Arg Tyr Asn Leu Asn 1 5 10 15 Phe HisGlu Ala Arg Gln Ala Cys Leu Asp Gln Asp Ala Val Ile Ala 20 25 30 Ser PheAsp Gln Leu Tyr Asp Ala Trp Arg Gly Gly Leu Asp Trp Cys 35 40 45 Asn AlaGly Trp Leu Ser Asp Gly Ser Val Gln Tyr Pro Ile Thr Lys 50 55 60 Pro ArgGlu Pro Cys Gly Gly Gln Asn Thr Val Pro Gly Val Arg Asn 65 70 75 80 TyrGly Phe Trp Asp Lys Asp Lys Ser Arg Tyr Asp Val Phe Cys Phe 85 90 95 ThrSer 97 amino acids amino acid single linear peptide 5 Gly Val Val PheHis Tyr Arg Pro Gly Ser Thr Arg Tyr Ser Leu Thr 1 5 10 15 Phe Glu GluAla Gln Asp Ala Cys Ile Arg Thr Gly Ala Ala Ile Ser 20 25 30 Ser Pro GluGln Leu Gln Ala Ala Tyr Ala Gly Tyr Glu Gln Cys Asp 35 40 45 Ala Gly TrpLeu Gln Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro 50 55 60 Arg Thr ProCys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Ile 65 70 75 80 Tyr GlyVal Arg Pro Ser Ser Glu Thr Tyr Asp Val Tyr Cys Tyr Val 85 90 95 Pro 101amino acids amino acid single linear peptide 6 Gly Gly Ser Ile Pro IlePro Gln Lys Leu Phe Gly Glu Val Thr Ser 1 5 10 15 Pro Leu Phe Pro LysPro Tyr Pro Asn Asn Phe Glu Thr Thr Thr Val 20 25 30 Ile Thr Val Pro ThrGly Tyr Arg Val Lys Leu Val Phe Gln Gln Phe 35 40 45 Asp Leu Glu Pro SerGlu Gly Cys Phe Tyr Asp Tyr Val Lys Ile Ser 50 55 60 Ala Asp Lys Lys SerLeu Gly Arg Phe Cys Gly Gln Leu Gly Ser Pro 65 70 75 80 Leu Gly Asn ProPro Gly Lys Lys Glu Phe Met Ser Gln Gly Asn Lys 85 90 95 Met Leu Leu ThrPhe 100 12 amino acids amino acid single linear peptide 7 Ala Val LeuPro Gln Glu Glu Glu Gly Xaa Gly Gly 1 5 10 5 amino acids amino acidsingle linear peptide 8 Ser Leu Pro Glu Gly 1 5 10 amino acids aminoacid single linear peptide 9 Ala Val Leu Pro Gln Glu Glu Glu Gly Ser 1 510 10 amino acids amino acid single linear peptide 10 Ser Leu Pro GlyGlu Ser Glu Glu Met Met 1 5 10 9 amino acids amino acid single linearpeptide 11 Val Xaa Xaa Asp Pro His Phe Ile Ile 1 5

What is claimed is:
 1. An isolated tumor necrosis factor-induced proteinor glycoprotein molecule TSG-6, or a functional derivative thereof,wherein said molecule has a sequence which substantially corresponds toat least 10 amino acids of SEQ ID NO:2.
 2. The molecule of claim 1having an apparent molecular weight of about 32 kDa under denaturingconditions.
 3. The molecule of claim 1 having an apparent molecularweight of 36 kDa to about 41 kDa under denaturing conditions.
 4. Themolecule of claim 1 having an apparent molecular weight of greater thanor equal to about 100 kDa.
 5. The molecule of claim 1 having the aminoacid sequence SEQ ID NO:2, or a functional derivative thereof.